Bispecific her2 and cd3 binding molecules

ABSTRACT

Provided herein are compositions, methods, and uses involving bispecific binding molecules that specifically bind to HER2, a receptor tyrosine kinase, and to CD3, a T cell receptor, and mediate T cell cytotoxicity for managing and treating disorders, such as cancer. Also provided herein are uses and methods for managing and treating HER2-related cancers.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/451,155, filed on Jan. 27, 2017, which isincorporated by reference herein in its entirety.

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled“Sequence_Listing_13542_041_999.txt” created on Jan. 25, 2018 and havinga size of 183 kbytes.

1. FIELD

Provided herein are compositions, methods, and uses involving bispecificbinding molecules that specifically bind to HER2, a receptor tyrosinekinase, and to CD3, a T cell receptor, and mediate T cell cytotoxicityfor managing and treating disorders, such as cancer.

2. BACKGROUND

HER2 is a receptor tyrosine kinase of the epidermal growth factorreceptor family. Amplification or overexpression of HER2 has beendemonstrated in the development and progression of cancers. Herceptin®(trastuzumab) is an anti-HER2 monoclonal antibody approved for treatingHER2-positive metastatic breast cancer and HER2-positive gastric cancer(Trastuzumab [Highlights of Prescribing Information]. South SanFrancisco, Calif.: Genentech, Inc.; 2014). Ertumaxomab is a tri-specificHER2-CD3 antibody with intact Fc-receptor binding (see, for example,Kiewe et al. 2006, Clin Cancer Res, 12(10): 3085-3091). Ertumaxomab is arat-mouse antibody; therefore, upon administration to humans, a humananti-mouse antibody response and a human anti-rat antibody response areexpected. 2502A, the parental antibody of ertumaxomab, has low affinityfor HER2 and low avidity (Diermeier-Daucher et al., MAbs, 2012, 4(5):614-622). There is a need for therapies capable of mediating T cellcytotoxicity in HER2-positive cancers.

3. SUMMARY

In a specific embodiment, provided herein is a method of treating aHER2-positive cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of abispecific binding molecule comprising an aglycosylated monoclonalantibody that is an immunoglobulin that binds to HER2, saidimmunoglobulin comprising two identical heavy chains and two identicallight chains, said light chains being a first light chain and a secondlight chain, wherein the first light chain is fused to a first singlechain variable fragment (scFv), via a peptide linker, to create a firstlight chain fusion polypeptide, and wherein the second light chain isfused to a second scFv, via a peptide linker, to create a second lightchain fusion polypeptide, wherein the first and second scFv (i) areidentical, and (ii) bind to CD3, and wherein the first and second lightchain fusion polypeptides are identical, and wherein the cancerexpresses a low level of HER2, and preferably wherein the cancer is nota head and neck cancer.

In a specific embodiment, the cancer is deemed to express a low level ofHER2 when the cancer expresses a lower level of HER2 than the level ofHER2 expressed by cancers that are indicated for treatment withtrastuzumab and are of the same tissue type as the HER2-positive cancer.

In a specific embodiment, the cancer is deemed to express a low level ofHER2 when the cancer has been determined not to overexpress HER2 basedon the following characterization of the cancer: (a) a firstdetermination of a level of HER2 in a test specimen comprising cells ofthe cancer is reported as negative, or (b) a first determination of alevel of HER2 in a test specimen comprising cells of the cancer isreported as equivocal, and a second determination of a level of HER2 ina test specimen comprising cells of the cancer is reported as equivocalor negative. In a specific embodiment, the determination of the level ofHER2 in the test specimen is reported as negative when the level of HER2in the test specimen is characterized as (i)(1) immunohistochemistry(IHC) 1+, wherein the level of HER2 in the test specimen ischaracterized as IHC 1+ when the test specimen exhibits an incompleteHER2 membrane staining that is faint/barely perceptible and withingreater than 10%/6 of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; (2) IHC 0, wherein thelevel of HER2 in the test specimen is characterized as IHC 0 when thetest specimen exhibits no HER2 staining observed, wherein the lack ofstaining is readily appreciated using a low-power objective, or a HER2membrane staining that is incomplete and is faint/barely perceptible andwithin less than or equal to 10% of the invasive tumor cells, whereinthe staining is readily appreciated using a low-power objective; or (ii)in situ hybridization (ISH) negative, wherein the level of HER2 in thetest specimen is characterized as ISH negative when the test specimenexhibits (1) a single-probe average HER2 copy number of less than 4.0signals per cell; or (2) a dual-probe HER2/CEP17 ratio of less than 2.0with an average HER2 copy number of less than 4.0 signals per cell. In aspecific embodiment, the determination of the level of HER2 in the testspecimen is reported as equivocal when the level of HER2 in the testspecimen is characterized as: (i) IHC 2+, wherein the level of HER2 inthe test specimen is characterized as IHC 2+ when the test specimenexhibits (1) a circumferential HER2 membrane staining that is incompleteand/or weak/moderate and within greater than 10% of invasive tumorcells, wherein the staining is observed in a homogenous and contiguouspopulation, and wherein the staining is readily appreciated using alow-power objective; or (2) a complete and circumferential HER2 membranestaining that is intense and within less than or equal to 10% ofinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; or (ii) ISH equivocal, wherein the level of HER2in the test specimen is characterized as ISH equivocal when the testspecimen exhibits (1) a single-probe ISH average HER2 copy number ofgreater than or equal to 4.0 and less than 6.0 signals/cell, wherein thecopy number is determined by counting at least 20 cells within the areaand is observed in a homogenous and contiguous population; or (2) adual-probe HER2/CEP17 ratio of less than 2.0 with an average HER2 copynumber of greater than or equal to 4.0 and less than 6.0 signals percell, wherein the copy number is determined by counting at least 20cells within the area and is observed in a homogenous and contiguouspopulation.

In a specific embodiment, the cancer is deemed to express a low level ofHER2 when a level of HER2 in a test specimen comprising cells of thecancer is characterized as IHC 2+ or less according to applicableAmerican Society of Clinical Oncology/College of American Pathologistsguideline recommendations for human epidermal growth factor receptor 2testing in cancer. In a preferred embodiment, the cancer is deemed toexpress a low level of HER2 when a level of HER2 in a test specimencomprising cells of the cancer is characterized as IHC 2+ or lessaccording to applicable American Society of Clinical Oncology/College ofAmerican Pathologists guideline recommendations for human epidermalgrowth factor receptor 2 testing in breast cancer. In a specificembodiment, a level of HER2 in a test specimen comprising cells of thecancer is characterized as IHC 2+. In a specific embodiment, the levelof HER2 in the test specimen is characterized as IHC 2+ when the testspecimen exhibits (1) a circumferential HER2 membrane staining that isincomplete and/or weak/moderate and within greater than 10% of invasivetumor cells, wherein the staining is observed in a homogenous andcontiguous population, and wherein the staining is readily appreciatedusing a low-power objective; or (2) a complete and circumferential HER2membrane staining that is intense and within less than or equal to 10%of invasive tumor cells, wherein the staining is readily appreciatedusing a low-power objective. In a specific embodiment, a level of HER2in a test specimen comprising cells of the cancer is characterized asIHC 1+. In a specific embodiment, the level of HER2 in the test specimenis characterized as IHC 1+ when the test specimen exhibits an incompleteHER2 membrane staining that is faint/barely perceptible and withingreater than 10% of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective. In a specificembodiment, a level of HER2 in a test specimen comprising cells of thecancer is characterized as IHC 0. In a specific embodiment, the level ofHER2 in the test specimen is characterized as IHC 0 when the testspecimen exhibits no HER2 staining observed, wherein the lack ofstaining is readily appreciated using a low-power objective, or a HER2membrane staining that is incomplete and is faint/barely perceptible andwithin less than or equal to 10% of the invasive tumor cells, whereinthe staining is readily appreciated using a low-power objective.

In a specific embodiment, the HER2-positive cancer that expresses a lowlevel of HER2 is a programmed death-ligand 1 (PDL1)-positive cancer. Ina specific embodiment, the HER2-positive cancer overexpresses PDL1relative to expression of PDL1 in analogous noncancerous cells of thesame tissue type as the cancer. In a specific embodiment, theHER2-positive cancer is deemed to overexpress PDL1 when a test specimencomprising cells of the cancer expresses a detectable level of PDL1above background. In a specific embodiment, the cancer is resistant toPDL1 blockade with an anti-PDL1 therapy. In a specific embodiment, theanti-PDL1 therapy is an anti-PDL1 antibody. In a specific embodiment,the anti-PDL1 antibody is atezolizumab. In a specific embodiment, thecancer is resistant to programmed cell death protein 1 (PD1) blockadewith an anti-PD1 therapy. In a specific embodiment, the anti-PD1 therapyis an anti-PD1 antibody. In a specific embodiment, the anti-PD1 antibodyis pembrolizumab.

In a specific embodiment, the HER2-positive cancer that expresses a lowlevel of HER2 is breast cancer, gastric cancer, an osteosarcoma,desmoplastic small round cell cancer, ovarian cancer, prostate cancer,pancreatic cancer, glioblastoma multiforme, gastric junctionadenocarcinoma, gastroesophageal junction adenocarcinoma, cervicalcancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma,Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma. In a specificembodiment, the cancer is gastric cancer or breast cancer. In a specificembodiment of a method described herein, the HER2-positive cancer thatexpresses a low level of HER2 is a metastatic tumor. In a specificembodiment, the metastatic tumor is a peritoneal metastasis. In aspecific embodiment, the cancer is resistant to treatment withtrastuzumab, cetuximab, lapatinib, erlotinib, or any other smallmolecule or antibody that targets the HER family of receptors.

In a specific embodiment, also provided herein is a method of treating aHER2-positive cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of abispecific binding molecule comprising an aglycosylated monoclonalantibody that is an immunoglobulin that binds to HER2, saidimmunoglobulin comprising two identical heavy chains and two identicallight chains, said light chains being a first light chain and a secondlight chain, wherein the first light chain is fused to a first singlechain variable fragment (scFv), via a peptide linker, to create a firstlight chain fusion polypeptide, and wherein the second light chain isfused to a second scFv, via a peptide linker, to create a second lightchain fusion polypeptide, wherein the first and second scFv (i) areidentical, and (ii) bind to CD3, and wherein the first and second lightchain fusion polypeptides are identical, and wherein the cancer is notindicated for treatment with trastuzumab, and preferably wherein thecancer is not a head and neck cancer. In a specific embodiment, thecancer is determined not to be indicated for treatment with trastuzumabbased on the following characterization of the cancer: (a) a firstdetermination of a level of HER2 in a test specimen comprising cells ofthe cancer is reported as negative, or (b) a first determination of alevel of HER2 in a test specimen comprising cells of the cancer isreported as equivocal, and a second determination of a level of HER2 ina test specimen comprising cells of the cancer is reported as equivocalor negative. In a specific embodiment, the determination of the level ofHER2 in the test specimen is reported as negative when the level of HER2in the test specimen is characterized as (i) (1) immunohistochemistry(IHC) 1+, wherein the level of HER2 in the test specimen ischaracterized as IHC 1+ when the test specimen exhibits an incompleteHER2 membrane staining that is faint/barely perceptible and withingreater than 10% of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; (2) IHC 0, wherein thelevel of HER2 in the test specimen is characterized as IHC 0 when thetest specimen exhibits no HER2 staining observed, wherein the lack ofstaining is readily appreciated using a low-power objective, or a HER2membrane staining that is incomplete and is faint/barely perceptible andwithin less than or equal to 10% of the invasive tumor cells, whereinthe staining is readily appreciated using a low-power objective; or (ii)in situ hybridization (ISH) negative, wherein the level of HER2 in thetest specimen is characterized as ISH negative when the test specimenexhibits (1) a single-probe average HER2 copy number of less than 4.0signals per cell; or (2) a dual-probe HER2/CEP17 ratio of less than 2.0with an average HER2 copy number of less than 4.0 signals per cell. In aspecific embodiment, the determination of the level of HER2 in the testspecimen is reported as equivocal when the level of HER2 in the testspecimen is characterized as: (i) IHC 2+, wherein the level of HER2 inthe test specimen is characterized as IHC 2+ when the test specimenexhibits (1) a circumferential HER2 membrane staining that is incompleteand/or weak/moderate and within greater than 10% of invasive tumorcells, wherein the staining is observed in a homogenous and contiguouspopulation, and wherein the staining is readily appreciated using alow-power objective; or (2) a complete and circumferential HER2 membranestaining that is intense and within less than or equal to 10% ofinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; or (ii) ISH equivocal, wherein the level of HER2in the test specimen is characterized as ISH equivocal when the testspecimen exhibits (1) a single-probe ISH average HER2 copy number ofgreater than or equal to 4.0 and less than 6.0 signals/cell, wherein thecopy number is determined by counting at least 20 cells within the areaand is observed in a homogenous and contiguous population; or (2) adual-probe HER2/CEP17 ratio of less than 2.0 with an average HER2 copynumber of greater than or equal to 4.0 and less than 6.0 signals percell, wherein the copy number is determined by counting at least 20cells within the area and is observed in a homogenous and contiguouspopulation. In a specific embodiment, the HER2-positive cancer is aprogrammed death-ligand 1 (PDL1)-positive cancer. In a specificembodiment, the HER2-positive cancer overexpresses PDL1 relative toexpression of PDL1 in analogous noncancerous cells of the same tissuetype as the cancer. In a specific embodiment, the HER2-positive canceris deemed to overexpress PDL1 when a test specimen comprising cells ofthe cancer expresses a detectable level of PDL1 above background. In aspecific embodiment, the cancer is resistant to PDL1 blockade with ananti-PDL1 therapy. In a specific embodiment, the anti-PDL1 therapy is ananti-PDL1 antibody. In a specific embodiment, the anti-PDL1 antibody isatezolizumab. In a specific embodiment, the cancer is resistant toprogrammed cell death protein 1 (PD1) blockade with an anti-PD1 therapy.In a specific embodiment, the anti-PD1 therapy is an anti-PD1 antibody.In a specific embodiment, the anti-PD1 antibody is pembrolizumab. In aspecific embodiment, the HER2-positive that is not indicated fortreatment with trastuzumab is breast cancer, gastric cancer, anosteosarcoma, desmoplastic small round cell cancer, ovarian cancer,prostate cancer, pancreatic cancer, glioblastoma multiforme, gastricjunction adenocarcinoma, gastroesophageal junction adenocarcinoma,cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia,melanoma, Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma. In aspecific embodiment, the HER2-positive that is not indicated fortreatment with trastuzumab is gastric cancer or breast cancer. In aspecific embodiment, the HER2-positive that is not indicated fortreatment with trastuzumab is a metastatic tumor. In a specificembodiment, the metastatic tumor is a peritoneal metastasis. In aspecific embodiment, the cancer is resistant to treatment withtrastuzumab, cetuximab, lapatinib, erlotinib, or any other smallmolecule or antibody that targets the HER family of receptors.

Also provided herein is a method of treating a HER2-positive,PDL1-positive cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of abispecific binding molecule comprising an aglycosylated monoclonalantibody that is an immunoglobulin that binds to HER2, saidimmunoglobulin comprising two identical heavy chains and two identicallight chains, said light chains being a first light chain and a secondlight chain, wherein the first light chain is fused to a first singlechain variable fragment (scFv), via a peptide linker, to create a firstlight chain fusion polypeptide, and wherein the second light chain isfused to a second scFv, via a peptide linker, to create a second lightchain fusion polypeptide, wherein the first and second scFv (i) areidentical, and (ii) bind to CD3, and wherein the first and second lightchain fusion polypeptides are identical, wherein the cancer is resistantto PDL1 blockade with an anti-PDL1 therapy and/or is resistant to PD1blockade with an anti-PD1 therapy. In a specific embodiment, theHER2-positive cancer overexpresses PDL1 relative to expression of PDL1in analogous noncancerous cells of the same tissue type as the cancer.In a specific embodiment, the HER2-positive cancer is deemed tooverexpress PDL1 when a test specimen comprising cells of the cancerexpresses a detectable level of PDL1 above background. In a specificembodiment, the anti-PDL1 therapy is an anti-PDL1 antibody. In aspecific embodiment, the anti-PDL1 antibody is atezolizumab. In aspecific embodiment, the anti-PD1 therapy is an anti-PD1 antibody. In aspecific embodiment, the anti-PD1 antibody is pembrolizumab. In aspecific embodiment, the HER2-positive cancer is breast cancer, gastriccancer, an osteosarcoma, desmoplastic small round cell cancer, squamouscell carcinoma of head and neck cancer, ovarian cancer, prostate cancer,pancreatic cancer, glioblastoma multiforme, gastric junctionadenocarcinoma, gastroesophageal junction adenocarcinoma, cervicalcancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma,Ewing's sarcoma, rhabdomyosarcoma, or neuroblastoma. In a specificembodiment, the HER2-positive cancer is a metastatic tumor. In aspecific embodiment, the metastatic tumor is a peritoneal metastasis. Ina specific embodiment, the cancer is resistant to treatment withtrastuzumab, cetuximab, lapatinib, erlotinib, or any other smallmolecule or antibody that targets the HER family of receptors.

In a specific embodiment of a method described herein, the sequence ofeach heavy chain is any of SEQ ID NOs: 23, 27, 62 or 63. In a specificembodiment, the sequence of each light chain is SEQ ID NO: 25. In aspecific embodiment, the sequence of the peptide linker is any of SEQ IDNOs: 14 or 35-41. In a specific embodiment, the sequence of a V_(H)domain in the first scFv is any of SEQ ID NOs: 15, 17 or 64. In aspecific embodiment, the sequence of an intra-scFv peptide linkerbetween a V_(H) domain and a V_(L) domain in the first scFv is any ofSEQ ID NOs: 14 or 35-41. In a specific embodiment, the sequence of aV_(L) domain in the first scFv is any of SEQ ID NOs: 16 or 65. In aspecific embodiment, the sequence of the scFv is any of SEQ ID NOs: 19or 48-59. In a specific embodiment, the sequence of the first lightchain fusion polypeptide is any of SEQ ID NOs: 29, 34, 42-47, or 60. Ina specific embodiment, the sequence of each heavy chain is SEQ ID NO: 27and the sequence of each light chain is SEQ ID NO: 25. In a specificembodiment, the sequence of the scFv is SEQ ID NO: 19. In a specificembodiment, the peptide linker is 5-30, 5-25, 5-15, 10-30, 10-20, 10-15,15-30, or 15-25 amino acids in length. In a specific embodiment, thesequence of the peptide linker is SEQ ID NO: 14. In a specificembodiment, the sequence of the first light chain fusion polypeptide isSEQ ID NO: 60. In a specific embodiment, the sequence of the heavy chainis SEQ ID NO: 62 and the sequence of each light chain fusion polypeptideis SEQ ID NO: 60. In a specific embodiment, the sequence of the firstlight chain fusion polypeptide is SEQ ID NO: 47. In a specificembodiment, the sequence of the heavy chain is SEQ ID NO: 27 and thesequence of each light chain fusion polypeptide is SEQ ID NO: 47. In aspecific embodiment, the sequence of the first light chain fusionpolypeptide is SEQ ID NO: 29. In a specific embodiment, the sequence ofthe heavy chain is SEQ ID NO: 27 and the sequence of each light chainfusion polypeptide is SEQ ID NO: 29. In a specific embodiment, the K_(D)of the bispecific binding molecule is between 70 nM and 1 μM for CD3. Ina specific embodiment, the bispecific binding molecule does not bind anFc receptor in its soluble or cell-bound form. In a specific embodiment,the heavy chain has been mutated to destroy an N-linked glycosylationsite. In a specific embodiment, the heavy chain has an amino acidsubstitution to replace an asparagine that is an N-linked glycosylationsite, with an amino acid that does not function as a glycosylation site.In a specific embodiment, the heavy chain has been mutated to destroy aC1q binding site. In a specific embodiment, the bispecific bindingmolecule does not activate complement. In a specific embodiment, thescFv is disulfide stabilized.

In a specific embodiment of a method described herein, the administeringis intravenous. In a specific embodiment of a method described herein,the administering is intraperitoneal, intrathecal, intraventricular inthe brain, or intraparenchymal in the brain. In a specific embodiment ofa method described herein, the administering is performed in combinationwith multi-modality anthracycline-based therapy.

In a specific embodiment of a method described herein, the methodfurther comprises administering to the subject doxorubicin,cyclophosphamide, paclitaxel, docetaxel, and/or carboplatin. In aspecific embodiment of a method described herein, the method furthercomprises administering to the subject radiotherapy. In a specificembodiment of a method described herein, the method further comprisesadministering to the subject an agent that increases cellular HER2expression.

In a specific embodiment of a method described herein, the bispecificbinding molecule is not bound to a T cell during said administeringstep.

In a specific embodiment of a method described herein, the methodfurther comprises administering T cells to the subject. In a specificembodiment, the T cells are bound to molecules identical to saidbispecific binding molecule.

In a specific embodiment of a method described herein, the subject is ahuman. In a specific embodiment, the subject is a canine.

In a specific embodiment of a method described herein, the bispecificbinding molecule is contained in a pharmaceutical composition, whichpharmaceutical composition further comprises a pharmaceuticallyacceptable carrier.

Also provided herein is a method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a cell expressing a bispecificbinding molecule of the invention, wherein the cancer expresses a lowlevel of HER2, and preferably wherein the cancer is not a head and neckcancer. In a specific embodiment, the sequence of the heavy chain of thebispecific binding molecule is SEQ ID NO: 27. In a specific embodiment,the nucleotide sequence encoding the heavy chain of the bispecificbinding molecule is SEQ ID NO: 26.

Also provided herein is a method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject (a) atherapeutically effective amount of an ex vivo cell comprising a vectorcomprising (i) a first polynucleotide comprising nucleotide sequencesencoding a light chain fusion polypeptide comprising an immunoglobulinlight chain fused to a scFv, via a peptide linker, operably linked to afirst promoter, and (ii) a second polynucleotide encoding animmunoglobulin heavy chain that binds to HER2 operably linked to asecond promoter, wherein the light chain binds to HER2 and wherein thescFv binds to CD3, or (b) a therapeutically effective amount of an exvivo cell comprising a mixture of polynucleotides comprising (i) a firstpolynucleotide comprising nucleotide sequences encoding a light chainfusion polypeptide comprising an immunoglobulin light chain fused to ascFv, via a peptide linker, operably linked to a first promoter, and(ii) a second polynucleotide encoding an immunoglobulin heavy chain thatbinds to HER2 operably linked to a second promoter; and wherein thecancer expresses a low level of HER2, and wherein the cancer is not ahead and neck cancer. In a specific embodiment, the sequence of theheavy chain is SEQ ID NO: 27. In a specific embodiment, the nucleotidesequence encoding the heavy chain is SEQ ID NO: 26.

Also provided herein is a method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a cell expressing a bispecificbinding molecule of the invention, wherein the cancer is not indicatedfor treatment with trastuzumab, and preferably wherein the cancer is nota head and neck cancer. In a specific embodiment, the sequence of theheavy chain of the bispecific binding molecule is SEQ ID NO: 27. In aspecific embodiment, the nucleotide sequence encoding the heavy chain ofthe bispecific binding molecule is SEQ ID NO: 26.

Also provided herein is a method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject (a) atherapeutically effective amount of an ex vivo cell comprising a vectorcomprising (i) a first polynucleotide comprising nucleotide sequencesencoding a light chain fusion polypeptide comprising an immunoglobulinlight chain fused to a scFv, via a peptide linker, operably linked to afirst promoter, and (ii) a second polynucleotide encoding animmunoglobulin heavy chain that binds to HER2 operably linked to asecond promoter, wherein the light chain binds to HER2 and wherein thescFv binds to CD3, or (b) a therapeutically effective amount of an exvivo cell comprising a mixture of polynucleotides comprising (i) a firstpolynucleotide comprising nucleotide sequences encoding a light chainfusion polypeptide comprising an immunoglobulin light chain fused to ascFv, via a peptide linker, operably linked to a first promoter, and(ii) a second polynucleotide encoding an immunoglobulin heavy chain thatbinds to HER2 operably linked to a second promoter; and wherein thecancer is not indicated for treatment with trastuzumab, and wherein thecancer is not a head and neck cancer.

In certain embodiments, provided herein is a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, comprising two identical heavy chainsand two identical light chains, said light chains being a first lightchain and a second light chain, wherein the first light chain is fusedto a first single chain variable fragment (scFv), via a peptide linker,to create a first light chain fusion polypeptide, and wherein the secondlight chain is fused to a second scFv, via a peptide linker, to create asecond light chain fusion polypeptide, wherein the first and second scFv(i) are identical, and (ii) bind to CD3, and wherein the first andsecond light chain fusion polypeptides are identical.

In certain embodiments of the bispecific binding molecule, the sequenceof each heavy chain is any of SEQ ID NOs: 23 or 27. In certainembodiments of the bispecific binding molecule, the sequence of eachlight chain is SEQ ID NO: 25. In certain embodiments of the bispecificbinding molecule, the sequence of the peptide linker is SEQ ID NO: 14.In certain embodiments of the bispecific binding molecule, the sequenceof a V_(H) domain in the first scFv is any of SEQ ID NOs: 15 or 17. Incertain embodiments of the bispecific binding molecule, the sequence ofan intra-scFv peptide linker between a V_(H) domain and a V_(L) domainin the first scFv is of SEQ ID NO: 14. In certain embodiments of thebispecific binding molecule, the sequence of a V_(L) domain in the firstscFv is of SEQ ID NO: 16. In certain embodiments of the bispecificbinding molecule, the sequence of the scFv is SEQ ID NO: 19. In certainembodiments of the bispecific binding molecule, the sequence of thefirst light chain fusion polypeptide is SEQ ID NO: 29.

In certain embodiments of the bispecific binding molecule, the sequenceof each heavy chain is any of SEQ ID NOs: 23, 27, 62 or 63. In certainembodiments of the bispecific binding molecule, the sequence of eachlight chain is SEQ ID NO: 25. In certain embodiments of the bispecificbinding molecule, the sequence of the peptide linker is any of SEQ IDNOs: 14 or 35-41. In certain embodiments of the bispecific bindingmolecule, the sequence of a V_(H) domain in the first scFv is any of SEQID NOs: 15, 17 or 64. In certain embodiments of the bispecific bindingmolecule, the sequence of an intra-scFv peptide linker between a V_(H)domain and a V_(L) domain in the first scFv is any of SEQ ID NOs: 14 or35-41. In certain embodiments of the bispecific binding molecule, thesequence of a V_(L) domain in the first scFv is any of SEQ ID NOs: 16 or65. In certain embodiments of the bispecific binding molecule, thesequence of the scFv is any of SEQ ID NOs: 19 or 48-59. In certainembodiments of the bispecific binding molecule, the sequence of thefirst light chain fusion polypeptide is any of SEQ ID NOs: 29, 34,42-47, or 60.

In certain embodiments of the bispecific binding molecule, the sequenceof each heavy chain is SEQ ID NO: 27 and the sequence of each lightchain is SEQ ID NO: 25. In certain embodiments of the bispecific bindingmolecule, the sequence of the scFv is SEQ ID NO: 19. In certainembodiments of the bispecific binding molecule, the sequence of theheavy chain is SEQ ID NO: 27, the sequence of each light chain is SEQ IDNO: 25 and the sequence of the scFv is SEQ ID NO: 19. In certainembodiments of the bispecific binding molecule, the peptide linker is5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acids inlength. In certain embodiments, the sequence of the peptide linker isSEQ ID NO: 14.

In certain embodiments, the sequence of the first light chain fusionpolypeptide is SEQ ID NO: 60. In certain embodiments, the sequence ofthe heavy chain is SEQ ID NO: 62 and the sequence of each light chainfusion polypeptide is SEQ ID NO: 60.

In certain embodiments, the sequence of the first light chain fusionpolypeptide is SEQ ID NO: 47. In certain embodiments, the sequence ofthe heavy chain is SEQ ID NO: 27 and the sequence of each light chainfusion polypeptide is SEQ ID NO: 47.

In certain embodiments, the sequence of the first light chain fusionpolypeptide is SEQ ID NO: 29. In certain embodiments, the sequence ofthe heavy chain is SEQ ID NO: 27 and the sequence of each light chainfusion polypeptide is SEQ ID NO: 29.

In certain embodiments of the bispecific binding molecule, the K_(D) isbetween 70 nM and 1 μM for CD3.

In certain embodiments of the bispecific binding molecule, the scFv ofthe bispecific binding molecule comprises one or more mutations tostabilize disulfide binding. In certain embodiments of the bispecificbinding molecule, the stabilization of disulfide binding preventsaggregation of the bispecific binding molecule. In certain embodimentsof the bispecific binding molecule, the stabilization of disulfidebinding reduces aggregation of the bispecific binding molecule ascompared to aggregation of the bispecific binding molecule without thestabilization of disulfide binding. In certain embodiments of thebispecific binding molecule, the one or more mutations to stabilizedisulfide binding comprise a V_(H) G44C mutation and a V_(L) Q100Cmutation (e.g., as present in SEQ ID NOS: 54-59). In certain embodimentsof the bispecific binding molecule, the one or more mutations tostabilize disulfide binding are the replacement of the amino acidresidue at V_(H)44 (according to the Kabat numbering system) with acysteine and the replacement of the amino acid residue at V_(L)100(according to the Kabat numbering system) with a cysteine so as tointroduce a disulfide bond between V_(H)44 and V_(L)100 (e.g., aspresent in SEQ ID NOS: 54-59).

In certain embodiments of the bispecific binding molecule, thebispecific binding molecule does not bind an Fc receptor in its solubleor cell-bound form. In certain embodiments of the bispecific bindingmolecule, the heavy chain has been mutated to destroy an N-linkedglycosylation site. In certain embodiments of the bispecific bindingmolecule, the heavy chain has an amino acid substitution to replace anasparagine that is an N-linked glycosylation site, with an amino acidthat does not function as a glycosylation site. In certain embodimentsof the bispecific binding molecule, the heavy chain has been mutated todestroy a C1q binding site. In certain embodiments, the bispecificbinding molecule does not activate complement.

In certain embodiments, provided herein is a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, comprising two identical heavy chainsand two identical light chains, said light chains being a first lightchain and a second light chain, wherein the first light chain is fusedto a first single chain variable fragment (scFv), via a peptide linker,to create a first light chain fusion polypeptide, and wherein the secondlight chain is fused to a second scFv, via a peptide linker, to create asecond light chain fusion polypeptide, wherein the first and second scFv(i) are identical, and (ii) bind to CD3, wherein the first and secondlight chain fusion polypeptides are identical, and wherein (a) thesequence of each heavy chain is SEQ ID NO: 62; and (b) the sequence ofeach light chain fusion polypeptide is SEQ ID NO: 60.

In certain embodiments, provided herein is a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, comprising two identical heavy chainsand two identical light chains, said light chains being a first lightchain and a second light chain, wherein the first light chain is fusedto a first single chain variable fragment (scFv), via a peptide linker,to create a first light chain fusion polypeptide, and wherein the secondlight chain is fused to a second scFv, via a peptide linker, to create asecond light chain fusion polypeptide, wherein the first and second scFv(i) are identical, and (ii) bind to CD3, wherein the first and secondlight chain fusion polypeptides are identical, and wherein (a) thesequence of each heavy chain is SEQ ID NO: 27; and (b) the sequence ofeach light chain fusion polypeptide is SEQ ID NO: 47.

In certain embodiments, provided herein is a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, comprising two identical heavy chainsand two identical light chains, said light chains being a first lightchain and a second light chain, wherein the first light chain is fusedto a first single chain variable fragment (scFv), via a peptide linker,to create a first light chain fusion polypeptide, and wherein the secondlight chain is fused to a second scFv, via a peptide linker, to create asecond light chain fusion polypeptide, wherein the first and second scFv(i) are identical, and (ii) bind to CD3, wherein the first and secondlight chain fusion polypeptides are identical, and wherein (a) thesequence of each heavy chain is SEQ ID NO: 27; and (b) the sequence ofeach light chain fusion polypeptide is SEQ ID NO: 29.

In certain embodiments, provided herein is a polynucleotide comprisingnucleotide sequences encoding a light chain fusion polypeptidecomprising an immunoglobulin light chain fused to a scFv, via a peptidelinker, wherein the light chain binds to HER2 and wherein the scFv bindsto CD3. In certain embodiments of the polynucleotide, the sequence ofthe light chain is SEQ ID NO: 25. In certain embodiments of thepolynucleotide, the nucleotide sequence encoding the light chain is SEQID NO: 24. In certain embodiments of the polynucleotide, the sequence ofthe scFv is SEQ ID NO: 19. In certain embodiments of the polynucleotide,the nucleotide sequence encoding the scFv is SEQ ID NO: 18. In certainembodiments of the polynucleotide, the sequence of the light chain isSEQ ID NO: 25 and the sequence of the scFv is SEQ ID NO: 19. In certainembodiments of the polynucleotide, the nucleotide sequence encoding thelight chain is SEQ ID NO: 24 and the nucleotide sequence encoding thescFv is SEQ ID NO: 18. In certain embodiments of the polynucleotide, thepeptide linker is 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25amino acids in length. In certain embodiments of the polynucleotide, thesequence of the peptide linker is SEQ ID NO: 14. In certain embodimentsof the polynucleotide, the nucleotide sequence encoding the peptidelinker is SEQ ID NO: 13.

In certain embodiments, provided herein is a vector comprising apolynucleotide encoding nucleotide sequences encoding a light chainfusion polypeptide comprising an immunoglobulin light chain fused to ascFv, via a peptide linker, wherein the light chain binds to HER2 andwherein the scFv binds to CD3, operably linked to a promoter. In certainembodiments, provided herein is an ex vivo cell comprising thepolynucleotide provided herein operably linked to a promoter. In certainembodiments, provided herein is an ex vivo cell comprising the vector.

In certain embodiments, provided herein is a vector comprising (i) afirst polynucleotide comprising nucleotide sequences encoding a lightchain fusion polypeptide comprising an immunoglobulin light chain fusedto a scFv, via a peptide linker, wherein the light chain binds to HER2and wherein the scFv binds to CD3 operably linked to a first promoter,and (ii) a second polynucleotide encoding an immunoglobulin heavy chainthat binds to HER2 operably linked to a second promoter. In certainembodiments, provided herein is an ex vivo cell comprising the vector.

In certain embodiments, provided herein is a method of producing abispecific binding molecule comprising (a) culturing the cell comprisingthe vector comprising (i) a first polynucleotide comprising nucleotidesequences encoding a light chain fusion polypeptide comprising animmunoglobulin light chain fused to a scFv, via a peptide linker,wherein the light chain binds to HER2 and wherein the scFv binds to CD3operably linked to a first promoter, and (ii) a second polynucleotideencoding an immunoglobulin heavy chain that binds to HER2 operablylinked to a second promoter, to express the first and secondpolynucleotides such that a bispecific binding molecule comprising saidlight chain fusion polypeptide and said immunoglobulin heavy chain isexpressed, and (b) recovering the bispecific binding molecule.

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of (i) the firstpolynucleotide operably linked to the first promoter, and (ii) thesecond polynucleotide encoding an immunoglobulin heavy chain that bindsto HER2 operably linked to the second promoter. In certain embodiments,provided herein is a pharmaceutical composition comprising atherapeutically effective amount of a vector comprising (i) the firstpolynucleotide operably linked to the first promoter, and (ii) thesecond polynucleotide encoding an immunoglobulin heavy chain that bindsto HER2 operably linked to the second promoter. In certain embodiments,the vector is a viral vector.

In certain embodiments, provided herein is a mixture of polynucleotidescomprising (i) a polynucleotide comprising nucleotide sequences encodinga light chain fusion polypeptide comprising an immunoglobulin lightchain fused to a scFv, via a peptide linker, wherein the light chainbinds to HER2 and wherein the scFv binds to CD3 operably linked to afirst promoter, and (ii) a second polynucleotide encoding animmunoglobulin heavy chain that binds to HER2 operably linked to asecond promoter. In certain embodiments of the mixture of polypeptides,the sequence of the heavy chain is SEQ ID NO: 27. In certain embodimentsof the mixture of polypeptides, the nucleotide sequence encoding theheavy chain is SEQ ID NO: 26. In certain embodiments, provided herein isan ex vivo cell comprising the mixture of polynucleotides providedherein.

In certain embodiments, provided herein is a method of producing abispecific binding molecule, comprising (i) culturing the cellcomprising the mixture of polynucleotides to express the first andsecond polynucleotides such that a bispecific binding moleculecomprising said light chain fusion polypeptide and said immunoglobulinheavy chain is produced, and (ii) recovering the bispecific bindingmolecule.

In certain embodiments, provided herein is a method of producing abispecific binding molecule, comprising (i) expressing the mixture ofpolynucleotides such that a bispecific binding molecule comprising saidfirst light chain fusion polypeptide and said immunoglobulin heavy chainis produced, and (ii) recovering the bispecific binding molecule.

In certain embodiments, provided herein is a method of making atherapeutic T cell comprising binding a bispecific binding moleculedescribed herein to a T cell. In certain embodiments, the T cell is ahuman T cell. In certain embodiments, the binding is noncovalently.

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the bispecific bindingmolecule and a pharmaceutically acceptable carrier.

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of the bispecific bindingmolecule, a pharmaceutically acceptable carrier, and T cells. In certainembodiments, the T cells are bound to the bispecific binding molecule.In certain embodiments, the binding of the T cells to the bispecificbinding molecule is noncovalently. In certain embodiments, the T cellsare administered to a subject for treatment of a HER2-positive cancer inthe subject. In certain embodiments, the T cells are autologous to thesubject to whom they are administered. In certain embodiments, the Tcells are allogeneic to the subject to whom they are administered. Incertain embodiments, the T cells are human T cells.

In certain embodiments, provided herein is a method of treating aHER2-positive cancer in a subject in need thereof comprisingadministering a pharmaceutical composition provided herein. In certainembodiments, provided herein is a method of treating a HER2-positivecancer in a subject in need thereof comprising administering atherapeutically effective amount of a bispecific binding moleculeprovided herein. In certain embodiments, the HER2-positive cancer isbreast cancer, gastric cancer, an osteosarcoma, desmoplastic small roundcell cancer, squamous cell carcinoma of head and neck cancer, ovariancancer, prostate cancer, pancreatic cancer, glioblastoma multiforme,gastric junction adenocarcinoma, gastroesophageal junctionadenocarcinoma, cervical cancer, salivary gland cancer, soft tissuesarcoma, leukemia, melanoma, Ewing's sarcoma, rhamdomyosarcoma,neuroblastoma, small cell lung cancer, or any other neoplastic tissuethat expresses the HER2 receptor. In certain embodiments, theHER2-positive cancer is a primary tumor or a metastatic tumor, e.g., abrain or peritoneal metastases.

In certain embodiments of the method of treating, the administering isintravenous. In certain embodiments of the method of treating, theadministering is intraperitoneal, intrathecal, intraventricular, orintraparenchymal. In certain embodiments of the method of treating, themethod further comprises administering to the subject doxorubicin,cyclophosphamide, paclitaxel, docetaxel, and/or carboplatin. In certainembodiments of the method of treating, the method further comprisesadministering to the subject radiotherapy. In certain embodiments of themethod of treating, the administering is performed in combination withmulti-modality anthracycline-based therapy. In certain embodiments ofthe method of treating, the administering is performed in combinationwith cytoreductive chemotherapy. In a specific embodiment, theadministering is performed after treating the subject with cytoreductivechemotherapy. In certain embodiments of the method of treating, thebispecific binding molecule is not bound to a T cell. In certainembodiments of the method of treating, the bispecific binding moleculeis bound to a T cell. In certain embodiments of the method of treating,the binding of the bispecific binding molecule to the T cell isnon-covalently. In certain embodiments of the method of treating, theadministering is performed in combination with T cell infusion. In aspecific embodiment, the administering is performed after treating thepatient with T cell infusion. In certain embodiments, the T cellinfusion is performed with T cells that are autologous to the patient towhom the T cells are administered. In certain embodiments, the T cellinfusion is performed with T cells that are allogeneic to the patient towhom the T cells are administered. In certain embodiments, the T cellscan be bound to molecules identical to a bispecific binding molecule asdescribed herein. In certain embodiments, the binding of the T cells tothe molecules identical to a bispecific binding molecule isnoncovalently. In certain embodiments, the T cells are human T cells.

In certain embodiments of the method of treating, the method furthercomprises administering to the subject an agent that increases cellularHER2 expression. In certain embodiments of the method of treating, theHER2-positive cancer is resistant to treatment with trastuzumab,cetuximab, lapatinib, erlotinib, or any other small molecule or antibodythat targets the HER family of receptors. In certain embodiments of themethod of treating, the subject is a human. In certain embodiments ofthe method of treating, the subject is a canine.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and FIG. 1E describe HER2-BsAb. FIG.1A depicts a schematic of the HER2-BsAb. The arrow points to the N297Amutation introduced into the heavy chain to remove glycosylation. FIG.1B depicts the purity of HER2-BsAb as demonstrated under reducingSDS-PAGE conditions. FIG. 1C depicts the purity of HER2-BsAb asdemonstrated by SEC-HPLC. FIG. 1D demonstrates that the N297A mutationin the human IgG1-Fc inhibits binding to the CD16A Fc receptor. FIG. 1Edemonstrates that the N297A mutation in the human IgG1-Fc inhibitsbinding to the CD32A Fc receptor.

FIG. 2A and FIG. 2B demonstrate that HER2-BsAb binds to a breast cancercell line and to T cells. FIG. 2A depicts the staining of AU565 breastcancer cells with trastuzumab (left) or with HER2-BsAb (right). FIG. 2Bdepicts the staining of CD3+ T cells with huOKT3 (left) or withHER2-BsAb (right).

FIG. 3 demonstrates that HER2-BsAb displays potent cytotoxic Tlymphocyte activity in a 4 hour ⁵¹Cr release assay. For a description oftrastuzumab-mOKT3, see, Thakur et al., 2010, Curr Opin Mol Ther, 12:340.

FIG. 4 compares the HER2 expression against HER2-BsAb T cellcytotoxicity in a panel of cancer cell lines.

FIG. 5A and FIG. 5B demonstrate that HER2-BsAb-redirected T cellcytotoxicity is antigen specific. FIG. 5A demonstrates that HER2-BsAbmediates T cell cytotoxicity against the HER2-positive cell line, UM SCC47, but not the HER2-negative cell line HTB-132. FIG. 5B demonstratesthat huOKT3 and trastuzumab can block the ability of HER2-BsAb tomediate T cell cytotoxicity.

FIG. 6 demonstrates that HER2-BsAb detects low levels of HER2 bycomparing the HER2-BsAb mediated T cell cytotoxicity to the HER2threshold of detection by flow cytometry.

FIG. 7A, FIG. 7B, and FIG. 7C provide the specificity, affinity, andantiproliferative action of HER2-BsAb. FIG. 7A demonstrates thatpre-incubation of the HER2-positive SKOV3 ovarian carcinoma cell lineblocks binding of HER2-BsAb. FIG. 7B demonstrates that SKOV3 cellslabeled with dilutions of trastuzumab or with HER2-BsAb display similarcurves when mean fluorescence intensity (MFI) is plotted againstantibody concentration. FIG. 7C demonstrates the antiproliferativeaction of HER2-BsAb compared against trastuzumab in the trastuzumabsensitive breast cancer cell line SKBR3.

FIG. 8 demonstrates that HER2-BsAb is effective against squamous cellcarcinoma of the head and neck (SCCHN) cell lines. A panel of SCCHNcells were analyzed for HER2-BsAb-mediated cytotoxicity and EC50 andcompared to the expression level of HER2 in each cell line as determinedby flow cytometry and by qRT-PCR.

FIG. 9A, FIG. 9B and FIG. 9C. HER2-BsAb mediates T cell cytotoxicityagainst SCCHN resistant to other HER targeted therapies. FIG. 9Ademonstrates that the SCCHN cell line PCI-30 expresses EGFR and HER2.FIG. 9B demonstrates that PCI-30 cells are resistant to HER-targetedtherapies lapatinib, erlotinib, neratinib, trastuzumab, and cetuximab.FIG. 9C demonstrates that PCI-30 cells are sensitive to T cells in thepresence of HER2-BsAb. Data represents the average of three differentcytotoxicity assays.

FIG. 10 demonstrates that HER2-BsAb is effective against osteosarcomacell lines. A panel of osteosarcoma cell lines were analyzed forHER2-BsAb-mediated cytotoxicity and EC50 and compared to the expressionlevel of HER2 in each cell line as determined by flow cytometry and byqRT-PCR

FIG. 11A, FIG. 11B, and FIG. 11C demonstrate that HER2-BsAb is effectiveagainst osteosarcoma cell lines resistant to other targeted therapies.FIG. 11A demonstrates that the osteosarcoma cell line U2OS expressesEGFR and HER2. FIG. 11B demonstrates that USOS cells are resistant toHER-targeted therapies lapatinib, erlotinib, neratinib, trastuzumab, andcetuximab. FIG. 11C demonstrates that USOS cells are sensitive to Tcells in the presence of HER2-BsAb. Data represents the average of threedifferent cytotoxicity assays.

FIG. 12A, FIG. 12B, FIG. 12C and FIG. 12D demonstrate that HER2-BsAb iseffective against the HeLa cervical carcinoma cell line resistant toother targeted therapies. FIG. 12A demonstrates that HeLa cells expressEGFR and HER2. FIG. 12B demonstrates that HeLa cells are resistant toHER-targeted therapies lapatinib, erlotinib, neratinib, trastuzumab, andcetuximab. FIG. 12C demonstrates that HeLa cells are sensitive to Tcells in the presence of HER2-BsAb. Data represents the average of threedifferent cytotoxicity assays. FIG. 12D demonstrates that pre-treatmentwith lapatinib enhances HeLa sensitivity to HER2-BsAb.

FIG. 13 demonstrates that HER2-BsAb reduces tumor growth in vivo. FIG.13 demonstrates that HER2-BsAb protects against tumor progression inimplanted MCF7 breast cancer cells mixed with PBMCs.

FIG. 14 demonstrates that HER2-BsAb protects against tumor progressionin implanted HCC1954 breast cancer mixed with peripheral bloodmononuclear cells (PBMC) in vivo.

FIG. 15 demonstrates that HER2-BsAb protects against a metastatic modelof tumor progression induced by intravenous introduction ofluciferase-tagged MCF7 cells in vivo.

FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D demonstrate that HER2-BsAbblocks the metastatic tumor growth of luciferase-tagged MCF7 cells invivo. FIG. 16A represents mice without treatment. FIG. 16B representsmice treated with PBMC and HER2-C825. FIG. 16C represents mice treatedwith HER2-BsAb. FIG. 16D represents mice treated with PBMC andHER2-BsAb.

FIG. 17A, FIG. 17B, and FIG. 17C describe HER2-BsAb. FIG. 17A depicts aschematic of the HER2-BsAb. The arrow points to the N297A mutationintroduced into the heavy chain to remove glycosylation. FIG. 17Bdepicts the purity of HER2-BsAb as demonstrated under reducing SDS-PAGEconditions. FIG. 17C depicts the purity of HER2-BsAb as demonstrated bysize exclusion chromatography high performance liquid chromatography(SEC-HPLC).

FIG. 18A, FIG. 18B, and FIG. 18C demonstrate that HER2-BsAb has the samespecificity, similar affinity, and antiproliferative effects astrastuzumab.

FIG. 19A and FIG. 19B demonstrate that HER2-BsAb redirected T cellcytotoxicity is HER2-specific and dependent on CD3.

FIG. 20 depicts HER2 expression and half maximal effective concentration(EC50) in the presence of ATC and HER2-BsAb in 35 different cell linesfrom different tumor systems.

FIG. 21A, FIG. 21B, FIG. 21C, FIG. 21D, FIG. 21E, FIG. 21F, FIG. 21G,FIG. 21H, and FIG. 21I demonstrate that HER2-BsAb mediates cytotoxicresponses against carcinoma cell lines resistant to other HER-targetedtherapies.

FIG. 22 demonstrates that the EC50 of HER2-BsAb correlates with the HER2level of expression determined by flow-cytometry. pM=picomolar; MFI=meanfluorescence intensity.

FIG. 23A, FIG. 23B, and FIG. 23C demonstrates that HER2-BsAb mediates Tcell cytotoxicity against PD-L1-positive HCC1954 targets in a mannerthat is relatively insensitive to PD-1 blockade by pembrolizumab, evenwith PD-1 expression on effector T cells.

FIG. 24A and FIG. 24B demonstrates that HER2-BsAb mediates T cellcytotoxicity against PD-L1-positive HEK-293 targets in a manner that isrelatively insensitive to PD-1 expression on effector T cells. Thecytotoxicity is an average of 6 experiments.

FIG. 25A, FIG. 25B, FIG. 25C, and FIG. 25D demonstrate that HER2-BsAb iseffective against HER2-positive xenografts.

FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, and FIG. 26E demonstrate invitro characterization of HER2-BsAb. FIG. 26A: HER2-BsAb has the samespecificity as trastuzumab. Pre-Incubation of the HER2(+)high SKOV3cells with trastuzumab prevents HER2-BsAb binding. FIG. 26B: HER2-BsAband trastuzumab have similar avidity for SKOV3 cells. Mean fluorescenceintensity (“MFI”) was plotted against the antibody concentration. FIG.26C: HER2-BsAb maintained same anti-proliferative effects as trastuzumabagainst the trastuzumab-sensitive SKBR3 cells. FIG. 26D: HER2-BsAbmediates T-cell cytotoxicity against the HER2(+) MCF-7 cells but not theHER2(−) HTB-132 cells. FIG. 26E: Blocking of HER2 or CD3 by trastuzumabor huOKT3, abrogates HER2-BsAb T-cell cytotoxicity. HER2(+) SCCHN PCI-13cells were used in the cytotoxicity assay. For this experiment 0.1 μg/mLof HER2-BsAb with 10 μg/mL of the blocking antibodies were used.

FIG. 27A and FIG. 27B demonstrate HER2-BsAb binding to T cells andredirecting T-cell killing. FIG. 27A: FACS histograms of HER2-BsAbbinding to naïve T cells purified from fresh PBMC (left panel) or ATCs(right panel). Concentrations of BsAbs (μg/10⁶ cells) were recorded onthe top of the left histogram, and Rituxan was used as negative control(mean fluorescence intensity set at 5). FIG. 27B: HER2-BsAb redirectedT-cell killing of HER2(+) AU565 breast cancer cells by 4-hour ⁵¹Crrelease assay. BsAb was either mixed directly with T cells and AU565together (mixing), or pre-incubated with T cells/target first (T cellspre-armed or AU565 pre-targeted), and unbound BsAb washed off beforeadding the other cells. ATC-to-target ratio was 10:1. Data points areshown as Mean±SEM.

FIG. 28A, FIG. 28B, FIG. 28C, and FIG. 28D demonstrate that HER2-BsAbmediates cytotoxic responses against carcinoma cell lines resistant toother HER targeted therapies. FIG. 28A, FIG. 28B, and FIG. 28C: Threerepresentative cell lines were used for FACS assay (upper panel),proliferation assay (middle panel), and HER2-BsAb mediated CTL assay(lower panel): (FIG. 28A) SCCHN PCI-30, (FIG. 28B) breast carcinomaHCC-1954, and (FIG. 28C) osteosarcoma U2OS. FIG. 28D: HER2-BsAb EC50inversely correlates with level of HER2 expression. Each of the celllines used in a cytotoxicity assay (Table 9) was assayed at least twice.The EC50 was determined each time and averaged. These values (exceptthose beyond assay limit 5 nM) were compared to HER2 expression (MFI).

FIG. 29A and FIG. 29B demonstrate that HER2-BsAb-mediated in vitroT-cell cytotoxicity was relatively insensitive to PD-L1 expression onthe tumor targets or PD-1 expression on T cells. FIG. 29A: FACS analysisof PD-L1 expression in HCC1954 cells (left panel), of induced PD-1expression in ATCs (middle panel), and HER2-BsAb-mediated cytotoxicity(right panel). FIG. 29B: FACS analysis of PD-L1 expression in HEK-293cells (left panel), and HER2-BsAb mediated cytotoxicity using the ATCsas in FIG. 29A (middle panel). Mean+SEM (n=6).

FIG. 30A, FIG. 30B, FIG. 30C, FIG. 30D, and FIG. 30E demonstrate thatHER2-BsAb is effective against HER2(+) breast cancer cell linexenografts. Treatment schedules were marked on the figures, and doses ofBsAbs and effector cells were detailed in the Results of Section 6.3.3.Data shown as mean+SEM (n=5). FIG. 30A: intravenous (“i.v.”) tumor plusi.v. effector cells model: Bioluminescence changes of MCF7 breastcancers during treatment. FIG. 30B and FIG. 30C: subcutaneous (“s.c.”tumor plus s.c. effector cells (mixing) model: % tumor growth of MCF7(FIG. 30B), and tumor volume changes of HCC1954 (FIG. 30C). FIG. 30D:s.c. tumor plus i.v. effector cells model: tumor volume changes ofHCC1954. FIG. 30E: HCC1954 s.c. tumor model as in (FIG. 30D), withtreatments of one dose of PBMC (2×10⁷ cells i.v.) at day 14, and twodoses of BsAbs (100 μg i.v.) at day 12 and 15. Representative images(200× magnifications) of IHC staining of tumor sections collected 5 daysafter i.v. PBMC were shown.

FIG. 31A and FIG. 31B demonstrate that HER2-BsAb is effective againstHER2(+) ovarian cancer cell line xenografts. Treatment schedules weremarked on the figures, and doses of BsAbs and effector cells weredetailed in the Results of Section 6.3.3. Data shown as mean+SEM (n=4).FIG. 31A: intraperitoneal (“i.p.”) tumor plus i.p./i.v. effector cellsmodel: Bioluminescence changes of SKOV3-luc ovarian cancers duringtreatment. FIG. 31B: Representative bioluminescence images at thebeginning (Day 13) and ending (Day 34) of the treatment were shown.

FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, and FIG. 32E demonstrate thatHER2-BsAb is effective against HER2(+) PDXs. s.c. tumor plus i.v.effector cells model was used for PDXs. Treatment schedules were markedon the figures, and doses of BsAbs and effector cells were detailed inthe Results of Section 6.3.3. Data shown as mean+SEM (n=5). FIG. 32A:Tumor volume changes of EK gastric cancer PDX. FIG. 32B: IHC images ofCD3 staining from another experiment with similar setting as in FIG.32A. Representative images (200× magnifications) of IHC staining oftumor sections collected 36 days after i.v. PBMC were shown. FIG. 32C:IHC images (200× magnifications) of HER2 staining of control treatedtumor sections. FIG. 32D and FIG. 32E: Average tumor volume changes ofM37 breast cancer PDX (FIG. 32D), and tumor growth of 5 individual mouse(black thin line) and averages (black thick line) in each group (FIG.32E).

FIG. 33A and FIG. 33B demonstrate that HER2-BsAb binding to CD3 on Tcells was functionally monovalent. FIG. 33A: Cytokine release from naïveT cells induced by 16.7 nM HER2-BsAb when compared to bivalent huOKT3IgG and monovalent huOKT3 Fab, in the absence (left panel) or presence(right panel) of HER2(+) NCI-N87 gastric tumor cells. Cytokine releaselevel below detection level was assigned as 1 μg/ml. FIG. 33B: T cellproliferation stimulated by 67 nM of the related antibodies, in theabsence of tumor targets. T cells only (Control) as the negativecontrol. OD reading at 450 nm (AU) was shown. All data points are shownas Mean+SD.

FIG. 34 demonstrates that HER2-BsAb is effective against HER2(+) breastcancer cell line xenografts that express PDL1 but are resistant to PD1or PDL1 treatment. s.c. tumor plus i.v. effector cell model: tumorvolume changes of HCC1954. Data shown as mean+SEM (n=5). Treatmentschedules are marked on the figures. s.c. 5×10⁶ HCC1954 xenografts weretreated with i.v. PBMC (7.5×10⁶, once per week for 2 weeks), and i.v.HER2-BsAb, anti-PD1 Pembrolizumab, or anti-PDL1 Atezolizumab (100 ugeach, twice per week for 4 weeks). Tumors were completely eradicatedwith HER2-BsAb treatment, in contrast to no effect for treatment withPD1/PDL1 blockade (i.e., treatment with anti-PD1 Pembrolizumab oranti-PDL1 Atezolizumab).

5. DETAILED DESCRIPTION

Provided herein are bispecific binding molecules that bind to both HER2and CD3. Also provided herein are isolated nucleic acids(polynucleotides), such as complementary DNA (cDNA), encoding suchbispecific binding molecules or fragments thereof. Further provided arevectors (e.g., expression vectors) and cells (e.g., ex vivo cells)comprising nucleic acids (polynucleotides) or vectors (e.g., expressionvectors) encoding such bispecific binding molecules or fragmentsthereof. Also provided herein are methods of making such bispecificbinding molecules, cells, and vectors. Also provided herein are T cellsbound to bispecific binding molecules provided herein. Also providedherein are methods of binding such bispecific binding molecules to Tcells. In other embodiments, provided herein are methods and uses fortreating HER2-positive cancers using the bispecific binding molecules,nucleic acids, vectors, and/or T cells described herein. Additionally,related compositions (e.g., pharmaceutical compositions), kits, anddiagnostic methods are also provided herein.

In certain embodiments, provided herein are bispecific binding moleculesthat specifically bind to HER2 and to CD3, and invoke T cellcytotoxicity for treating cancer. Without being bound by any theory, itis believed that the bispecific binding molecules described herein notonly bind tumors to T cells, they also cross-link CD3 on T cells andinitiate the activation cascade, and, this way, T cell receptor(TCR)-based cytotoxicity is redirected to desired tumor targets,bypassing major histocompatibility complex (MHC) restrictions.

5.1 Bispecific Binding Molecules

Provided herein are bispecific binding molecules that bind to HER2 andCD3. A binding molecule, which can be used within the methods providedherein, is a bispecific binding molecule comprising an aglycosylatedmonoclonal antibody that is an immunoglobulin that binds to HER2,comprising two identical heavy chains and two identical light chains,said light chains being a first light chain and a second light chain,wherein the first light chain is fused to a first single chain variablefragment (scFv), via a peptide linker, to create a first fusionpolypeptide, and wherein the second light chain is fused to a secondscFv, via a peptide linker, to create a second fusion polypeptide,wherein the first and second scFv (i) are identical, and (ii) bind toCD3, and wherein the first and second fusion polypeptides are identical.

HER2 is a member of the epidermal growth factor receptor (EGFR) familyof receptor tyrosine kinases. In a specific embodiment, HER2 is humanHER2. GenBank™ accession number NM_004448.3 (SEQ ID NO: 1) provides anexemplary human HER2 nucleic acid sequence. GenBank™ accession numberNP_004439.2 (SEQ ID NO: 2) provides an exemplary human HER2 amino acidsequence. In another specific embodiment, HER2 is canine HER2. GenBank™accession number NM_001003217.1 (SEQ ID NO: 3) provides an exemplarycanine HER2 nucleic acid sequence. GenBank™ accession numberNP_001003217.1 (SEQ ID NO: 4) provides an exemplary canine HER2 aminoacid sequence.

CD3 is a T cell co-receptor comprised of a gamma chain, a delta chain,and two epsilon chains. In a specific embodiment, CD3 is a human CD3.GenBank™ accession number NM_000073.2 (SEQ ID NO: 5) provides anexemplary human CD3 gamma nucleic acid sequence. GenBank™ accessionnumber NP_000064.1 (SEQ ID NO: 6) provides an exemplary human CD3 gammaamino acid sequence. GenBank™ accession number NM_000732.4 (SEQ ID NO:7) provides an exemplary human CD3 delta nucleic acid sequence. GenBank™accession number NP_000723.1 (SEQ ID NO: 8) provides an exemplary humanCD3 delta amino acid sequence. GenBank™ accession number NM_000733.3(SEQ ID NO: 9) provides an exemplary human CD3 epsilon nucleic acidsequence. GenBank™ accession number NP_000724.1 (SEQ ID NO: 10) providesan exemplary human CD3 epsilon amino acid sequence. In another specificembodiment, CD3 is a canine CD3. GenBank™ accession numberNM_001003379.1 (SEQ ID NO: 11) provides an exemplary canine CD3 epsilonnucleic acid sequence. GenBank™ accession number NP_001003379.1 (SEQ IDNO: 12) provides an exemplary canine CD3 epsilon amino acid sequence.

The immunoglobulin in the bispecific binding molecules of the inventioncan be, as non-limiting examples, a monoclonal antibody, a nakedantibody, a chimeric antibody, a humanized antibody, or a humanantibody. As used herein, the term “immunoglobulin” is used consistentwith its well known meaning in the art, and comprises two heavy chainsand two light chains. Methods for making antibodies are described inSection 5.3.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity-determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as,for example, horse, monkey, cow, pig, cat, or dog.

A humanized antibody is an antibody produced by recombinant DNAtechnology, in which some or all of the amino acids of a humanimmunoglobulin light or heavy chain that are not required for antigenbinding (e.g., the constant regions and the framework regions of thevariable domains) are used to substitute for the corresponding aminoacids from the light or heavy chain of the cognate, nonhuman antibody.By way of example, a humanized version of a murine antibody to a givenantigen has on both of its heavy and light chains (1) constant regionsof a human antibody; (2) framework regions from the variable domains ofa human antibody; and (3) CDRs from the murine antibody. When necessary,one or more residues in the human framework regions can be changed toresidues at the corresponding positions in the murine antibody so as topreserve the binding affinity of the humanized antibody to the antigen.This change is sometimes called “back mutation.” Similarly, forwardmutations may be made to revert back to murine sequence for a desiredreason, e.g., stability or affinity to antigen. Without being bound byany theory, humanized antibodies generally are less likely to elicit animmune response in humans as compared to chimeric human antibodiesbecause the former contain considerably fewer non-human components.

The term “epitope” is art-recognized and is generally understood bythose of skill in the art to refer to the region of an antigen thatinteracts with an antibody. An epitope of a protein antigen can belinear or conformational, or can be formed by contiguous ornoncontiguous amino acid sequences of the antigen.

A scFv is an art-recognized term. An scFv comprises a fusion protein ofthe variable regions of the heavy (V_(H)) and light (V_(L)) chains of animmunoglobulin, wherein the fusion protein retains the same antigenspecificity as the whole immunoglobulin. The V_(H) is fused to the V_(L)via a peptide linker (such a peptide linker is sometimes referred toherein as an “intra-scFv peptide linker”).

In certain embodiments of the invention, the scFv has a peptide linkerthat is between 5-30, 5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25amino acid residues in length. In certain embodiments, the scFv peptidelinker displays one or more characteristics suitable for a peptidelinker known to one of ordinary skill in the art. In certainembodiments, the scFv peptide linker comprises amino acids that allowfor scFv peptide linker solubility, such as, for example, serine andthreonine. In certain embodiments, the scFv peptide linker comprisesamino acids that allow for scFv peptide linker flexibility, such as, forexample, glycine. In certain embodiments, the scFv peptide linkerconnects the N-terminus of the V_(H) to the C-terminus of the V_(L). Incertain embodiments, the scFv peptide linker can connect the C-terminusof the V_(H) to the N-terminus of the V_(L). In certain embodiments, thescFv peptide linker is a linker as described in Table 1, below (e.g.,any one of SEQ ID NOs: 14, or 35-41). In a preferred embodiment, thepeptide linker is SEQ ID NO: 14.

In certain embodiments of the bispecific binding molecules of theinvention, the scFv that binds to CD3 comprises the V_(H) and the V_(L)of a CD3-specific antibody known in the art, such as, for example,huOKT3 (see, for example, Adair et al., 1994, Hum Antibodies Hybridomas5:41-47), YTH12.5 (see, for example Routledge et al., 1991, Eur JImmunol, 21: 2717-2725), HUM291 (see, for example, Norman et al., 2000,Clinical Transplantation, 70(12): 1707-1712), teplizumab (see, forexample, Herold et al., 2009, Clin Immunol, 132: 166-173), huCLB-T3/4(see, for example, Labrijn et al., 2013, Proceedings of the NationalAcademy of Sciences, 110(13): 5145-5150), otelixizumab (see, forexample, Keymeulen et al., 2010, Diabetologia, 53: 614-623),blinatumomab (see, for example, Cheadle, 2006, Curr Opin Mol Ther, 8(1):62-68), MT110 (see, for example, Silke and Gires, 2011, MAbs, 3(1):31-37), catumaxomab (see, for example, Heiss and Murawa, 2010, Int JCancer, 127(9): 2209-2221), 28F111 (see, for example, Canadian PatentApplication CA 2569509 A1), 27H5 (see, for example, Canadian PatentApplication CA 2569509 A1), 23F10 (see, for example, Canadian PatentApplication CA 2569509 A1), 15C3 (see, for example, Canadian PatentApplication CA 2569509 A1), visilizumab (see, for example, Dean et al.,2012, Swiss Med Wkly, 142: w13711), and Hum291 (see, for example, Deanet al., 2012, Swiss Med Wkly, 142: wl3711).

In certain embodiments, the scFv in a bispecific binding molecule of theinvention binds to the same epitope as a CD3-specific antibody known inthe art. In a specific embodiment, the scFv in a bispecific bindingmolecule of the invention binds to the same epitope as the CD3-specificantibody huOKT3. Binding to the same epitope can be determined by assaysknown to one skilled in the art, such as, for example, mutationalanalyses or crystallographic studies. In certain embodiments, the scFvcompetes for binding to CD3 with an antibody known in the art. In aspecific embodiment, the scFv in a bispecific binding molecule of theinvention competes for binding to CD3 with the CD3-specific antibodyhuOKT3. Competition for binding to CD3 can be determined by assays knownto one skilled in the art, such as, for example, flow cytometry. See,for example, Section 6.1.2.4. In certain embodiments, the scFv comprisesa V_(H) with at least 85%, 90%, 95%, 98%, or at least 99% similarity tothe V_(H) of a CD3-specific antibody known in the art. In certainembodiments, the scFv comprises the V_(H) of a CD3-specific antibodyknown in the art, comprising between 1 and 5 conservative amino acidsubstitutions. In certain embodiments, the scFv comprises a V_(L) withat least 85%, 90%, 95%, 98%, or at least 99% similarity to the V_(L) ofa CD3-specific antibody known in the art. In certain embodiments, thescFv comprises the V_(L) of a CD3-specific antibody known in the art,comprising between 1 and 5 conservative amino acid substitutions.

Conservative amino acid substitutions are amino acid substitutions thatoccur within a family of amino acids, wherein the amino acids arerelated in their side chains. Generally, genetically encoded amino acidsare divided into families: (1) acidic, comprising aspartate andglutamate; (2) basic, comprising arginine, lysine, and histidine; (3)non-polar, comprising isoleucine, alanine, valine, proline, methionine,leucine, phenylalanine, tryptophan; and (4) uncharged polar, comprisingcysteine, threonine, glutamine, glycine, asparagine, serine, andtyrosine. In addition, an aliphatic-hydroxy family comprises serine andthreonine. In addition, an amide-containing family comprises asparagineand glutamine. In addition, an aliphatic family comprises alanine,valine, leucine and isoleucine. In addition, an aromatic familycomprises phenylalanine, tryptophan, and tyrosine. Finally, asulfur-containing side chain family comprises cysteine and methionine.As an example, one skilled in the art would reasonably expect anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Preferred conservative amino acid substitutiongroups include: lysine-arginine, alanine-valine, phenylalanine-tyrosine,glutamic acid-aspartic acid, valine-leucine-isoleucine,cysteine-methionine, and asparagine-glutamine.

In a preferred embodiment, the scFv is derived from the huOKT3 antibody,and thus contains the V_(H) and V_(L) of huOKT3 monoclonal antibody (SEQID NOS: 15 and 16, respectively). See, for example, Van Wauwe et al.,1991, nature, 349: 293-299. In specific embodiments of the bispecificbinding molecule, the scFv is derived from the huOKT3 monoclonalantibody and has no more than 5 amino acid mutations relative to nativehuOKT3 V_(H) and V_(L) sequences. In certain embodiments of thebispecific binding molecule, the scFv is derived from the huOKT3monoclonal antibody and comprises one or more mutations, relative tonative huOKT3 V_(H) and V_(L) sequences, to stabilize disulfide binding.In certain embodiments of the bispecific binding molecule, thestabilization of disulfide binding prevents aggregation of thebispecific binding molecule. In certain embodiments of the bispecificbinding molecule, the stabilization of disulfide binding reducesaggregation of the bispecific binding molecule as compared toaggregation of the bispecific binding molecule without the stabilizationof disulfide binding. In certain embodiments of the bispecific bindingmolecule, the one or more mutations to stabilize disulfide bindingcomprise a V_(H) G44C mutation and a V_(L) Q100C mutation (e.g., aspresent in SEQ ID NOS: 54-59). In certain embodiments of the bispecificbinding molecule, the one or more mutations to stabilize disulfidebinding are the replacement of the amino acid residue at V_(H)44(according to the Kabat numbering system) with a cysteine and thereplacement of the amino acid residue at V_(L)100 (according to theKabat numbering system) with a cysteine so as to introduce a disulfidebond between V_(H)44 and V_(L)100 (e.g., as present in SEQ ID NOS:54-59). In an especially preferred embodiment, the scFv comprises theV_(H) of huOKT3 comprising the amino acid substitution at numberedposition 105, wherein the cysteine is substituted with a serine (SEQ IDNO: 17). In certain embodiments, the sequence of the V_(H) of the scFvis as described in Table 4, below (e.g., any one of SEQ ID NOs: 15, 17,or 64). In certain embodiments, the sequence of the V_(L) of the scFv isas described in Table 5, below (e.g., any one of SEQ ID NOs: 16 or 65).In certain embodiments, the sequence of the scFv is as described inTable 6, below (e.g., any one of SEQ ID NOs: 19 or 48-59). In apreferred embodiment, the sequence of the scFv is SEQ ID NO: 19. In aspecific embodiment, the scFv comprises a variant of the V_(H) of huOKT3that has no more than 5 amino acid mutations relative to the nativesequence of huOKT3 V_(H). In a specific embodiment, the scFv comprises avariant of the V_(L) of huOKT3 that has no more than 5 amino acidmutations relative to the native sequence of huOKT3 V_(L).

The sequences of the variable regions of an anti-CD3 scFv may bemodified by insertions, substitutions and deletions to the extent thatthe resulting scFv maintains the ability to bind to CD3, as determinedby, for example, ELISA, flow cytometry, and BiaCore™. The ordinarilyskilled artisan can ascertain the maintenance of this activity byperforming the functional assays as described herein below, such as, forexample, binding analyses and cytotoxicity analyses.

In certain embodiments, the peptide linker conjugating theimmunoglobulin light chain and the scFv is between 5-30, 5-25, 5-15,10-30, 10-20, 10-15, 15-30, or 15-25 amino acids in length. In certainembodiments, the peptide linker displays one or more characteristicssuitable for a peptide linker known to one of ordinary skill in the art.In certain embodiments, the peptide linker comprises amino acids thatallow for peptide linker solubility, such as, for example, serine andthreonine. In certain embodiments, the peptide linker comprises aminoacids that allow for peptide linker flexibility, such as, for example,glycine. In certain embodiments, the sequence of the peptide linkerconjugating the immunoglobulin light chain and the scFv is as describedin Table 1, below (e.g., any one of SEQ ID NOs: 14 or 35-41). Inpreferred embodiments, the peptide linker is SEQ ID NO: 14.

In certain embodiments of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 comprises the heavychain and/or the light chain of a HER2-specific antibody known in theart, such as, for example, trastuzumab (see, for example, Baselga et al.1998, Cancer Res 58(13): 2825-2831), M-111 (see, for example, Higgins etal., 2011, J Clin Oncol, 29(Suppl): Abstract TPSI 19), pertuzumab (see,for example, Franklin et al., 2004, Cancer Cell, 5: 317-328),ertumaxomab (see, for example, Kiewe and Thiel, 2008, Expert OpinInvestig Drugs, 17(10): 1553-1558), MDXH210 (see, for example, Schwaabet al., 2001, Journal of Immunotherapy, 24(1): 79-87), 2B1 (see, forexample, Borghaei et al., 2007, J Immunother, 30: 455-467), and MM-302(see, for example, Wickham and Futch, 2012, Cancer Research, 72(24):Supplement 3). In certain embodiments of the bispecific bindingmolecules of the invention, the immunoglobulin that binds to HER2comprises the heavy chain of trastuzumab. In certain embodiments of thebispecific binding molecules of the invention, the immunoglobulin thatbinds to HER2 comprises the sequence as set forth in SEQ ID NO: 23. Incertain embodiments of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 comprises a variant ofthe heavy chain of trastuzumab (see, e.g., Table 2, below). In aspecific embodiment of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 comprises a variant ofthe light chain of trastuzumab that has no more than 5 amino acidmutations relative to the native sequence of trastuzumab. In certainembodiments of the bispeciftic binding molecules of the invention, theimmunoglobulin that binds to HER2 comprises the light chain oftrastuzumab (SEQ ID NO: 25). In certain embodiments of the bispecificbinding molecules of the invention, the immunoglobulin that binds toHER2 comprises a variant of the light chain of trastuzumab. In aspecific embodiment of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 comprises a variant ofthe light chain of trastuzumab that has no more than 5 amino acidmutations relative to the native sequence of trastuzumab.

In certain embodiments of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 binds to the sameepitope as a HER2-specific antibody known in the art. In a specificembodiment, the immunoglobulin in a bispecific binding molecule of theinvention binds to the same epitope as trastuzumab. Binding to the sameepitope can be determined by assays known to one skilled in the art,such as, for example, mutational analyses or crystallographic studies.In certain embodiments, the immunoglobulin that binds to HER2 competesfor binding to HER2 with an antibody known in the art. In a specificembodiment, the immunoglobulin in a bispecific binding molecule of theinvention competes for binding to HER2 with trastuzumab. Competition forbinding to HER2 can be determined by assays known to one skilled in theart, such as, for example, flow cytometry. See, for example, Section6.1.2.4. In certain embodiments, the immunoglobulin comprises a V_(H)with at least 85%, 90%, 95%, 98%, or at least 99% similarity to theV_(H) of a HER2-specific antibody known in the art. In certainembodiments, the immunoglobulin comprises the V_(H) of a HER2-specificantibody known in the art, comprising between 1 and 5 conservative aminoacid substitutions. In certain embodiments, the immunoglobulin comprisesa V_(L) with at least 85%, 90%, 95%, 98%, or at least 99% similarity tothe V of a HER2-specific antibody known in the art. In certainembodiments, the immunoglobulin comprises the V_(L) of a HER2-specificantibody known in the art, comprising between 1 and 5 conservative aminoacid substitutions. In certain embodiments, the immunoglobulin comprisesa V_(H) of a heavy chain described in Table 2, below (e.g., the V_(H) ofany one of SEQ ID NOs: 23, 27, 62, or 63). In certain embodiments, theimmunoglobulin comprises a V_(L) of a light chain described in Table 3,below (e.g., the V_(L) of SEQ ID NO: 25).

The sequences of the variable regions of an anti-HER2 antibody may bemodified by insertions, substitutions and deletions to the extent thatthe resulting antibody maintains the ability to bind to HER2, asdetermined by, for example, ELISA, flow cytometry, and BiaCore™. Theordinarily skilled artisan can ascertain the maintenance of thisactivity by performing the functional assays as described herein below,such as, for example, binding analyses and cytotoxicity analyses.

In certain embodiments of the bispecific binding molecules of theinvention, the immunoglobulin that binds to HER2 is an IgG1immunoglobulin.

Methods of producing human antibodies are known to one skilled in theart, such as, for example, phage display methods described above usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/60433, WO 98/24893, WO 98/16664, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety. The techniques of Cole et al., and Boerder etal., are also available for the preparation of human monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).

In certain embodiments, human antibodies are produced using transgenicmice, which are incapable of expressing functional endogenous mouseimmunoglobulins, but which can express human immunoglobulin genes. Forexample, the human heavy and light chain immunoglobulin gene complexesmay be introduced randomly or by homologous recombination into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells in addition to the human heavy and light chain genes. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. In particular,homozygous deletion of the JH region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, for example, all or a portion of a polypeptideprovided herein. Monoclonal antibodies directed against the antigen canbe obtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, for example, PCT publications WO98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,886,793; 5,916,771; and 5,939,598,which are incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm(San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engagedto provide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Human monoclonal antibodies can also be made by immunizing micetransplanted with human peripheral blood leukocytes, splenocytes or bonemarrows (e.g., Trioma techniques of XTL). Completely human antibodieswhich recognize a selected epitope can be generated using a techniquereferred to as “guided selection.” In this approach a selected non-humanmonoclonal antibody, for example, a mouse antibody, is used to guide theselection of a completely human antibody recognizing the same epitope.See, for example, Jespers et al., Bio/technology 12:899-903 (1988).Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

Methods for making humanized antibodies are known to one skilled in theart. See, for example, Winter EP 0 239 400; Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239: 1534-1536 (1988); Queen et al., Proc.Nat. Acad. ScL USA 86:10029 (1989); U.S. Pat. No. 6,180,370; and Orlandiet al., Proc. Natl. Acad. Sd. USA 86:3833 (1989); the disclosures of allof which are incorporated by reference herein in their entireties.Generally, the transplantation of murine (or other non-human) CDRs ontoa human antibody is achieved as follows. The cDNAs encoding heavy andlight chain variable domains are isolated from a hybridoma. The DNAsequences of the variable domains, including the CDRs, are determined bysequencing. The DNAs, encoding the CDRs are inserted into thecorresponding regions of a human antibody heavy or light chain variabledomain coding sequences, attached to human constant region gene segmentsof a desired isotype (e.g., gamma-1 for CH and K for C_(L)), are genesynthesized. The humanized heavy and light chain genes are co-expressedin mammalian host cells (e.g., CHO or NSO cells) to produce solublehumanized antibody. To facilitate large scale production of antibodies,it is often desirable select for high expressor using a DHFR gene or GSgene in the producer line. These producer cell lines are cultured inbioreactors, or hollow fiber culture system, or WAVE technology, toproduce bulk cultures of soluble antibody, or to produce transgenicmammals (e.g., goats, cows, or sheep) that express the antibody in milk(see, e.g., U.S. Pat. No. 5,827,690).

Antibody fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH, domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. In certainembodiments, elements of a human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A fully human antibody also can be constructed bygenetic or chromosomal transfection methods, as well as phage displaytechnology, all of which are known in the art. See, for example,McCafferty et al., Nature 348:552-553 (1990) for the production of humanantibodies and fragments thereof in vitro, from immunoglobulin variabledomain gene repertoires from unimmunized donors. In this technique,antibody variable domain genes are cloned in-frame into either a majoror minor coat protein gene of a filamentous bacteriophage, and displayedas functional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. In this way, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats, for their review, see e.g. Johnson and Chiswell, CurrentOpinion in Structural Biology 3:5564-571 (1993).

Antibody humanization can also be performed by, for example,synthesizing a combinatorial library comprising the six CDRs of anon-human target monoclonal antibody fused in frame to a pool ofindividual human frameworks. A human framework library that containsgenes representative of all known heavy and light chain human germlinegenes can be utilized. The resulting combinatorial libraries can then bescreened for binding to antigens of interest. This approach can allowfor the selection of the most favorable combinations of fully humanframeworks in terms of maintaining the binding activity to the parentalantibody. Humanized antibodies can then be further optimized by avariety of techniques.

Antibody humanization can be used to evolve mouse or other non-humanantibodies into “fully human” antibodies. The resulting antibodycontains only human sequence and no mouse or non-human antibodysequence, while maintaining similar binding affinity and specificity asthe starting antibody.

For full length antibody molecules, the immunoglobulin genes can beobtained from genomic DNA or mRNA of hybridoma cell lines. Antibodyheavy and light chains are cloned in a mammalian vector system. Assemblyis documented with double strand sequence analysis. The antibodyconstruct can be expressed in other human or mammalian host cell lines.The construct can then be validated by transient transfection assays andWestern blot analysis of the expressed antibody of interest. Stable celllines with the highest productivity can be isolated and screened usingrapid assay methods.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJ, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A), or thelike, or heteromylomas, fusion products thereof, or any cell or fusioncell derived therefrom, or any other suitable cell line as known in theart. See, for example, the ATCC or LifeTech website, and the like, withantibody producing cells, such as, but not limited to, isolated orcloned spleen, peripheral blood, lymph, tonsil, or other immune or Bcell containing cells, or any other cells expressing heavy or lightchain constant or variable or framework or CDR sequences, either asendogenous or heterologous nucleic acid, as recombinant or endogenous,viral, bacterial, algal, prokaryotic, amphibian, avian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See, forexample, Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

In a preferred specific embodiment, the bispecific binding moleculecomprises a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule does not bind or has reduced binding to an Fcreceptor (FcR), in soluble form or cell-bound form (including onimmune-effector cells, such as, for example, NK cells, monocytes, andneutrophils). These FcRs include, but are not limited to, FcRI (CD64),FcRII (CD32), and FcRIII (CD16). The affinity to FcR(n), the neonatal Fcreceptor, is not affected, and thus maintained in the bispecific bindingmolecule. For example, if the immunoglobulin is an IgG, preferably, theIgG has reduced or no affinity for an Fc gamma receptor. In certainembodiments, one or more positions within the Fc region that makes adirect contact with Fc gamma receptor, such as, for example, amino acids234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop, are mutatedsuch that the bispecific binding molecule has a decreased or no affinityfor an Fc gamma receptor. See, for example, Sondermann et al., 2000,Nature, 406: 267-273, which is incorporated herein by reference in itsentirety. Preferably, for an IgG, the mutation N297A is made to destroyFc receptor binding. In certain embodiments, affinity of the bispecificbinding molecule or fragment thereof for an Fc gamma receptor isdetermined by, for example, BiaCore™ assay, as described, for example,in Okazaki et al., 2004. J Mol Biol, 336(5):1239-49. See also, Section6. In certain embodiments, the bispecific binding molecule comprisingsuch a variant Fc region binds an Fc receptor on a FcR-bearingimmune-effector cell with less than 25%, 20%, 15%, 10%, or 5% binding ascompared to a reference Fc region. Without being bound by any particulartheory, a bispecific binding molecule comprising such a variant Fcregion will have a decreased ability to induce a cytokine storm. Inpreferred embodiments, the bispecific binding molecule comprising such avariant Fc region does not bind an Fc receptor in soluble form or as acell-bound form.

In certain embodiments, the bispecific binding molecule comprises avariant Fc region, such as, for example, an Fc region with additions,deletions, and/or substitutions to one or more amino acids in the Fcregion of an antibody provided herein in order to alter effectorfunction, or enhance or diminish affinity of antibody to FcR. In apreferred embodiment, the affinity of the antibody to FcR is diminished.Reduction or elimination of effector function is desirable in certaincases, such as, for example, in the case of antibodies whose mechanismof action involves blocking or antagonism but not killing of the cellsbearing a target antigen. In certain embodiments, the Fc variantsprovided herein may be combined with other Fc modifications, includingbut not limited to modifications that alter effector function. Incertain embodiments, such modifications provide additive, synergistic,or novel properties in antibodies or Fc fusions. Preferably, the Fcvariants provided herein enhance the phenotype of the modification withwhich they are combined.

In preferred embodiments, the bispecific binding molecule of theinvention is aglycosylated. Preferably, this is achieved by mutating theanti-HER2 immunoglobulin portion of the bispecific binding molecule inits Fc receptor to destroy a glycosylation site, preferably an N-linkedglycosylation site. In another specific embodiment, an immunoglobulin ismutated to destroy an N-linked glycosylation site. In certain preferredembodiments, the bispecific binding molecule has been mutated to destroyan N-linked glycosylation site. In certain embodiments, the heavy chainof the bispecific binding molecule has an amino acid substitution toreplace an asparagine that is an N-linked glycosylation site, with anamino acid that does not function as a glycosylation site. In apreferred embodiment, the method encompasses deleting the glycosylationsite of the Fc region of a bispecific binding molecule, by modifyingposition 297 from asparagine to alanine (N297A). For example, in certainembodiments, the bispecific binding molecule comprises a heavy chainwith the sequence of SEQ ID NO: 20. As used herein, “glycosylationsites” include any specific amino acid sequence in an antibody to whichan oligosaccharide (i.e., carbohydrates containing two or more simplesugars linked together) will specifically and covalently attach.Oligosaccharide side chains are typically linked to the backbone of anantibody via either N- or O-linkages. N-linked glycosylation refers tothe attachment of an oligosaccharide moiety to the side chain of anasparagine residue. O-linked glycosylation refers to the attachment ofan oligosaccharide moiety to a hydroxyamino acid, e.g., serine,threonine. Methods for modifying the glycosylation content of antibodiesare well known in the art, see, for example, U.S. Pat. No. 6,218,149; EP0 359 096 B1; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S.Publication No. 2003/0115614; U.S. Pat. Nos. 6,218,149; 6,472,511; allof which are incorporated herein by reference in their entirety. Inanother embodiment, aglycosylation of the bispecific binding moleculesof the invention can be achieved by recombinantly producing thebispecific binding molecule in a cell or expression system incapable ofglycosylation, such as, for example, bacteria. In another embodiment,aglycosylation of the bispecific binding molecules of the invention canbe achieved by enzymatically removing the carbohydrate moieties of theglycosylation site.

In preferred embodiments, the bispecific binding molecule of theinvention does not bind or has reduced binding affinity (relative to areference or wild type immunoglobulin) to the complement component C1q.Preferably, this is achieved by mutating the anti-HER2 immunoglobulinportion of the bispecific binding molecule to destroy a C1q bindingsite. In certain preferred embodiments, the method encompasses deletingthe C1q binding site of the Fc region of an antibody, by modifyingposition 322 from lysine to alanine (K322A). For example, in certainembodiments, the bispecific binding molecule comprises a heavy chainwith the sequence of SEQ ID NO: 21. In certain embodiments, affinity ofthe bispecific binding molecule or fragment thereof for the complementcomponent C1q is determined by, for example, BiaCore™ assay, asdescribed, for example, in Okazaki et al., 2004. J Mol Biol,336(5):1239-49. See also, Section 6. In certain embodiments, thebispecific binding comprising an anti-HER2-immunoglobulin comprising adestroyed C1q binding site binds the complement component C1q with lessthan 25%, 20%, 15%, 10%, or 5% binding compared to a reference or wildtype immunoglobulin. In certain embodiments, the bispecific bindingmolecule does not activate complement.

In preferred embodiments, the bispecific binding molecule of theinvention comprises an immunoglobulin, wherein the immunoglobulin (i)comprises at least one amino acid modification relative to a wild-typeFc region, such that said molecule does not bind or has reduced bindingto an Fc receptor in soluble form or as cell-bound form; (ii) comprisesone or more mutations in the Fc region to destroy an N-linkedglycosylation site; and (iii) does not or has reduced binding to thecomplement component C1q. For example, in certain embodiments, thebispecific binding molecule comprises an IgG comprising a firstmutation, N297A, in the Fc region to (i) abolish or reduce binding to anFc receptor in soluble form or as cell-bound form; and (ii) destroy anN-linked glycosylation site in the Fc region; and a second mutation,K322A, in the Fc region to (iii) abolish or reduce binding to thecomplement component C1q. See, for example, SEQ ID NO: 27.

In a preferred embodiment, the immunoglobulin that binds to HER2comprises the variable regions of trastuzumab (see, e.g., Tables 2 and3), and preferably a human IgG1 constant region. In a preferredembodiment, the immunoglobulin that binds to HER2 comprises the variableregions of trastuzumab wherein the sequence of the heavy chain is SEQ IDNO: 27 and wherein the sequence of the light chain is SEQ ID NO: 25. Ina preferred embodiment, the immunoglobulin that binds to HER2 is avariant of trastuzumab, wherein the heavy chain does not bind or hasreduced binding to an Fc receptor in soluble form or as cell-bound form.In a preferred embodiment, the heavy chain that does not bind an Fcreceptor in soluble form or as a cell-bound form comprises a mutation inthe Fc region to destroy an N-linked glycosylation site. In a preferredembodiment, the heavy chain has an amino acid substitution to replace anasparagine that is an N-linked glycosylation site, with an amino acidthat does not function as a glycosylation site. In a preferredembodiment, the mutation to destroy an N-linked glycosylation site isN297A in the Fc region (SEQ ID NO: 20). In a preferred embodiment, theimmunoglobulin that binds to HER2 comprises the variable regions oftrastuzumab, wherein the sequence of the heavy chain comprises amutation in the Fc region to destroy a C1q binding site. In a preferredembodiment, the immunoglobulin does not activate complement. In apreferred embodiment, the mutation to destroy a C1q binding site isK322A in the Fc region (SEQ ID NO: 21). In an especially preferredembodiment, the immunoglobulin that binds to HER2 comprises the variableregions of trastuzumab, wherein the immunoglobulin heavy chain comprisesa mutation in the Fc region to destroy an N-linked glycosylation siteand a mutation in the Fc region to destroy a C1q binding site (see, forexample, SEQ ID NO: 27). In an especially preferred embodiment, theimmunoglobulin that binds to HER2 comprises the variable regions oftrastuzumab wherein the sequence of the heavy chain of theimmunoglobulin has been mutated in the Fc region and is SEQ ID NO: 27and wherein the sequence of the light chain is SEQ ID NO: 25. In anespecially preferred embodiment, the sequence of the light chain fusionpolypeptide is SEQ ID NO: 29. In certain embodiments, the heavy chaincomprises the constant region of trastuzumab. In certain embodiments,the heavy chain comprises the constant region of a heavy chain describedin Table 2, below (e.g., the constant region of any one of SEQ ID NOs:23, 27, 62, or 63). In certain embodiments, the sequence of the heavychain is as described in Table 2, below (e.g., any one of SEQ ID NOs:23, 27, 62, or 63). In certain embodiments, the light chain comprisesthe constant region of a light chain described in Table 3, below (e.g.,the constant region of SEQ ID NO: 25). In certain embodiments, thesequence of the light chain is as described in Table 3, below (e.g., SEQID NO: 25).

In certain embodiments, the bispecific binding molecule has atrastuzumab-derived sequence that contains one or more of themodifications in the trastuzumab immunoglobulin, and has ahuOKT3-derived sequence that contains one or more of the modificationsin the huOKT3 V_(H) and V_(L) sequences, as described in Table 8, below.Bispecific binding molecules having other immunoglobulin or scFvsequences can contain analogous mutations at corresponding positions inthese other immunoglobulin or scFv sequences. In certain embodiments,the bispecific binding molecule is (a) derived from trastuzumab andhuOKT3; and (b) contains one or more of the modifications as describedin Table 8, below. In certain embodiments, the sequence of the peptidelinker conjugating the immunoglobulin light chain and the scFv is asdescribed in Table 1, below (e.g., any one of SEQ ID NOs: 14 or 35-41).In certain embodiments, the sequence of the heavy chain is as describedin Table 2, below (e.g., any one of SEQ ID NOs: 23, 27, 62, or 63). Incertain embodiments, the sequence of the light chain is as described inTable 3, below (e.g., SEQ ID NO: 25). In certain embodiments, thesequence of the V_(H) of the scFv is as described in Table 4, below(e.g., any one of SEQ ID NOs: 15, 17, or 64). In certain embodiments,the sequence of the V_(L) of the scFv is as described in Table 5, below(e.g., any one of SEQ ID NOs: 16 or 65). In certain embodiments, thesequence of the scFv peptide linker is as described in Table 1, below(e.g., any one of SEQ ID NOs: 14 or 35-41). In certain embodiments, thesequence of the scFv is as described in Table 6, below (e.g., any one ofSEQ ID NOs: 19 48-59, or 66). In certain embodiments, the sequence ofthe light chain fusion polypeptide is as described in Table 7, below(e.g., any one of SEQ ID NOs: 29, 34, 42-47, or 60).

In certain embodiments, the bispecific binding molecule comprises aglycosylated monoclonal antibody that is an immunoglobulin that binds toHER2, comprising two identical heavy chains and two identical lightchains, said light chains being a first light chain and a second lightchain, wherein the first light chain is fused to a first single chainvariable fragment (scFv), via a peptide linker, to create a first lightchain fusion polypeptide, and wherein the second light chain is fused toa second scFv, via a peptide linker, to create a second light chainfusion polypeptide, wherein the first and second scFv (i) are identical,and (ii) bind to CD3, wherein the first and second light chain fusionpolypeptides are identical, wherein the sequence of each heavy chain isSEQ ID NO: 62, and wherein the sequence of each light chain fusionpolypeptide is SEQ ID NO: 60.

In certain embodiments, the bispecific binding molecule comprises aglycosylated monoclonal antibody that is an immunoglobulin that binds toHER2, comprising two identical heavy chains and two identical lightchains, said light chains being a first light chain and a second lightchain, wherein the first light chain is fused to a first single chainvariable fragment (scFv), via a peptide linker, to create a first lightchain fusion polypeptide, and wherein the second light chain is fused toa second scFv, via a peptide linker, to create a second light chainfusion polypeptide, wherein the first and second scFv (i) are identical,and (ii) bind to CD3, wherein the first and second light chain fusionpolypeptides are identical, wherein the sequence of each heavy chain isSEQ ID NO: 27, and wherein the sequence of each light chain fusionpolypeptide is SEQ ID NO: 47.

In certain embodiments, the bispecific binding molecule comprises aglycosylated monoclonal antibody that is an immunoglobulin that binds toHER2, comprising two identical heavy chains and two identical lightchains, said light chains being a first light chain and a second lightchain, wherein the first light chain is fused to a first single chainvariable fragment (scFv), via a peptide linker, to create a first lightchain fusion polypeptide, and wherein the second light chain is fused toa second scFv, via a peptide linker, to create a second light chainfusion polypeptide, wherein the first and second scFv (i) are identical,and (ii) bind to CD3, wherein the first and second light chain fusionpolypeptides are identical, wherein the sequence of each heavy chain isSEQ ID NO: 27, and wherein the sequence of each light chain fusionpolypeptide is SEQ ID NO: 29.

In certain embodiments, the bispecific binding molecule has lowimmunogenicity. Low or acceptable immunogenicity and/or high affinity,as well as other suitable properties, can contribute to the therapeuticresults achieved. “Low immunogenicity” is defined herein as raisingsignificant HAHA, HACA or HAMA responses in less than about 75%, orpreferably less than about 50% of the patients treated and/or raisinglow titres in the patient treated (Elliott et al., Lancet 344:1125-1127(1994), entirely incorporated herein by reference).

The bispecific binding molecules provided herein can bind HER2 and CD3with a wide range of affinities. The affinity or avidity of an antibodyfor an antigen can be determined experimentally using any suitablemethod. See, for example, Berzofsky, et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company:New York, N.Y. (1992); and methods described herein. The measuredaffinity of a particular antibody-antigen interaction can vary ifmeasured under different conditions (e.g., salt concentration, pH).Thus, measurements of affinity and other antigen-binding parameters arepreferably made with standardized solutions of antibody and antigen, anda standardized buffer, such as the buffer described herein. Theaffinity, K_(D) is a ratio of k_(on)/k_(off). Generally, a K_(D) in themicromolar range is considered low affinity. Generally, a K_(D) in thepicomolar range is considered high affinity. In another specificembodiment, the bispecific binding molecule has high affinity for HER2and low affinity for CD3. In another specific embodiment, the bispecificbinding molecule has high affinity for HER2 and average affinity forCD3. In a specific embodiment, the bispecific binding molecule has aK_(D) of between 70 nM and 1 μM for CD3. In a specific embodiment, thebispecific binding molecule has a K_(D) of between 70 nM and 500 nM forCD3. In a specific embodiment, the bispecific binding molecule has aK_(D) of between 500 nM and 1 JIM for CD3.

In certain embodiments, the bispecific binding molecule binds to one ormore HER2-positive carcinoma cell lines, as determined by assays knownto one skilled in the art, such as, for example, ELISA, BiaCore™, andflow cytometry. In certain embodiments, the carcinoma cell line is abreast carcinoma cell line, such as, for example, MDA-MB-361,MDA-MB-468, AU565, SKBR3, HTB27, HTB26, HCC1954, and/or MCF7. In certainembodiments, the carcinoma cell line is an ovarian carcinoma cell line,such as, for example, OVCAR3 and/or SKOV3. In certain embodiments, thecarcinoma cell line is a gastric carcinoma cell line, such as, forexample, NCI-N87, KATO III, AGS, and/or SNU-16. In certain embodiments,the carcinoma cell line is a melanoma cell line, such as, for example,HT144, SKMEL28, M14, and/or HTB63. In certain embodiments, the carcinomacell line is an osteosarcoma cell line, such as, for example, RG160,RG164, CRL1427, and/or U2OS. In certain embodiments, the carcinoma cellline is a Ewings sarcoma cell line, such as, for example, SKEAW and/orSKES-1. In certain embodiments, the carcinoma cell line is arhabdomyosarcoma cell line, such as, for example, HTB82. In certainembodiments, the carcinoma cell line is a neuroblastoma cell line, suchas, for example, NMB7, SKNBE(2)C, IMR32, SKNBE(2)S, SKNBE(1)N, and/orNB5. In certain embodiments, the carcinoma cell line is a squamous cellcarcinoma head and neck (SCCHN) cell lines, such as, for example, 15B,93-VU-147T, PCI-30, UD-SCC2, PCI-15B, SCC90, and/or UMSCC47. In certainembodiments, the carcinoma cell line is a cervical cancer cell line,such as, for example, HeLa. In certain embodiments, the carcinoma cellline is a small cell lung cancer cell line, such as, for example,NCI-H524, NCI-H69, and/or NCI-H345. In certain embodiments, thebispecific binding molecule binds to the HER2-positive carcinoma cellline with an EC50 in the picomolar range. See, for example, Section6.1.3.4 and Section 6.1.3.6.

In certain embodiments, the bispecific binding molecule binds to CD3+ Tcells, as determined by assays known to one skilled in the art, such as,for example, ELISA, BiaCore™, and flow cytometry. In certain preferredembodiments, the bispecific binding molecule binds to CD3+ T cells withgreater than 15-fold less binding than huOKT3 binding to CD3+ T cells.See, for example, Section 6.1.3.1. In certain embodiments, the CD3+ Tcells are human T cells.

In certain embodiments, the bispecific binding molecule the bispecificbinding molecule mediates T cell cytotoxicity against HER2-positivecells, as determined by assays known to one skilled in the art, such as,for example, cytotoxicity assays. In preferred embodiments, thebispecific binding molecule mediates T cell cytotoxicity againstHER2-positive cell lines with an EC50 in the picomolar range. In certainembodiments, the HER2-positive cells are breast carcinoma cell lines,such as, for example, MDA-MB-361, MDA-MB-468, AU565, SKBR3, HTB27,HTB26, and/or MCF7. In certain embodiments, the HER2-positive cells areof an ovarian carcinoma cell line, such as, for example, OVCAR3 and/orSKOV3. In certain embodiments, the HER-2 positive cells are of a gastriccarcinoma cell line, such as, for example, NCI-N87, KATO III, AGS,and/or SNU-16. In certain embodiments, the HER2-positive cells are of amelanoma cell line, such as, for example, HT144, SKMEL28, M14, and/orHTB63. In certain embodiments, the HER2-positive cells are of anosteosarcoma cell line, such as, for example, RG160, RG164, CRL1427,and/or U2OS. In certain embodiments, the HER2-positive cells are of anEwings sarcoma cell line, such as, for example, SKEAW and/or SKES-1. Incertain embodiments, the HER2-positive cells are of a rhabdomyosarcomacell line, such as, for example, HTB82. In certain embodiments, theHER2-positive cells are of a neuroblastoma cell line, such as, forexample, NMB7, SKNBE(2)C, IMR32, SKNBE(2)S, SKNBE(1)N, and/or NB5. Incertain embodiments, the HER2-positive cells are of a squamous cellcarcinoma head and neck (SCCHN) cell line, such as, for example, 15B,93-VU-147T, PCI-30, UD-SCC2, PCI-15B, SCC90, and/or UMSCC47. In certainembodiments, the HER2-positive cells are of a cervical cancer cell line,such as, for example, HeLa. In certain embodiments, the HER2-positivecells are of a small cell lung cancer cell line, such as, for example,NCI-H524, NCI-H69, and/or NCI-H345. See, for example, Section 6.1.3.4and Section 6.1.3.6.

In certain embodiments, preincubation of HER2-positive cells with huOKT3blocks the ability of the bispecific binding molecule to induce T cellcytotoxicity. In certain embodiments, preincubation of HER2-positivecells with trastuzumab blocks the ability of the bispecific bindingmolecule to induce T cell cytotoxicity. See, for example, Section6.1.3.3.

In certain embodiments, the bispecific binding molecule mediates T cellcytotoxicity against HER2-positive cells, wherein the level ofHER2-expression in said cells is below the threshold of detection byflow cytometry performed with the bispecific binding molecule. See, forexample, Section 6.1.3.4.

In certain embodiments, the bispecific binding molecule mediates T cellcytotoxicity against HER2-positive cells resistant to other HER-targetedtherapies, such as, for example, trastuzumab, cetuximab, lapatinib,erlotinib, neratinib, or any other small molecule or antibody thattargets the HER family of receptors. In a specific embodiment, the tumorthat is resistant to HER-targeted therapies, such as, for example,trastuzumab, cetuximab, lapatinib, erlotinib, neratinib, or any othersmall molecule or antibody that targets the HER family of receptors isresponsive to treatment with a bispecific binding molecule to theinvention. See, for example, Section 6.1.3.7, Section 6.1.3.8, Section6.1.3.9, and Section 6.1.3.10.

In certain embodiments, the bispecific binding molecule reducesHER2-positive tumor progression, metastasis, and/or tumor size. See, forexample, Section 6.1.3.11.

In certain embodiments, the bispecific binding molecule is bound to a Tcell. In certain embodiments, the binding of the bispecific bindingmolecule to a T cell is noncovalently. In certain embodiments, the Tcell is administered to a subject. In certain embodiments, the T cell isautologous to the subject to whom the T cell is to be administered. Incertain embodiments, the T cell is allogeneic to the subject to whom theT cell is to be administered. In certain embodiments, the T cell is ahuman T cell.

In certain embodiments, the bispecific binding molecule is not bound toa T cell.

In certain embodiments, the bispecific binding molecule is conjugated toan organic moiety, a detectable marker, and/or isotope as described inSection 5.2.

In certain embodiments, the bispecific binding molecule or fragmentthereof is produced as described in Section 5.3. In certain embodiments,the bispecific binding molecule or fragment thereof is encoded by apolynucleotide as described in Section 5.3.1. In certain embodiments,the bispecific binding molecule or fragment thereof is encoded by avector (e.g., expression vector) as described in Section 5.3.2. Incertain embodiments, the bispecific binding molecule or fragment thereofis produced from a cell as described in Section 5.3.2.

In certain embodiments, the bispecific binding molecule is a componentof a composition (e.g., pharmaceutical composition) and/or as part of akit as described in Section 5.5.

In certain embodiments, the bispecific binding molecule is usedaccording to the methods provided in Section 5.6. In certainembodiments, the bispecific binding molecule is used as a diagnostictool according to the methods provided in Section 5.6.2. In certainembodiments, the bispecific binding molecule is used as a therapeuticaccording to the methods provided in Section 5.6.1. In certainembodiments, the bispecific binding molecule is administered to asubject, such as a subject described in Section 5.7, for use accordingto the methods provided in Section 5.6. In certain embodiments, thebispecific binding molecule is administered to a subject as part of acombination therapy as described in Section 5.9, for use according tothe methods provided in Section 5.6.

TABLE 1 Linker Sequence DESCRIPTION SEQUENCE (SEQ ID NO:) (G₄S)₃GGGGSGGGGSGGGGS (SEQ ID NO: 14) TS(G₄S)₃ LinkerTSGGGGSGGGGSGGGGS (SEQ ID NO: 35) G₄S Linker GGGGS (SEQ ID NO: 36)(G₄S₎₂ Linker GGGGSGGGGS (SEQ ID NO: 37) (G₄S)₃ LinkerGGGGSGGGGSGGGGS (SEQ ID NO: 38) (G₄S)₄ LinkerGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 39) (G₄S)₅ LinkerGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 40) (G₄S)₆ LinkerGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41)

Table 2. Heavy Chain Sequence. The non-italicized, non-underlinedsequence represents the V_(H) domain. The italicized sequence representsthe constant region. The underlined, italicized, and bold sequencesrepresent the mutations described in the “DESCRIPTION” column.

DESCRIPTION SEQUENCE (SEQ ID NO:) Trastuzumab V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG domain with humanLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA IgG1 constant regionEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23) Trastuzumab V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG domain with humanLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA IgG1 constantEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL region; N297A;APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS K322ASGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVLHQDWLNGKEY KC

VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) Trastuzumab V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG domain with humanLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA IgG1 constantEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL region; N297AAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQY

STYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62) Trastuzumab V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG domain with humanLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA IgG1 constantEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL region; K322AAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KC

VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62)

TABLE 3 Light Chain Sequence.The non-italicized sequence represents the V_(L) domain. The italicizedsequence represents the constant region. DESCRIPTIONSEQUENCE (SEQ ID NO:) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chainKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTS (SEQ ID NO: 25)

TABLE 4 scFv V_(H) Sequence.The underlined, italicized, and bold sequences represent themutations described in the ″DESCRIPTION″ column. DESCRIPTIONSEQUENCE (SEQ ID NO:) huOKT3 V_(H)QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSS (SEQ ID NO: 15) huOKT3 V_(H); C105SQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP EDTGVYFCARYYDDHY

LDYWGQGTPVTVSS (SEQ ID NO: 17) huOKT3 V_(H); C105S +QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK V_(H)-G44C

LEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP EDTGVYFCARYYDDHY

LDYWGQGTPVTVSS (SEQ ID NO: 64)

TABLE 5 scFv V_(L) Sequence.The underlined, italicized, and bold sequence represent themutations described in the ″DESCRIPTION″ column. DESCRIPTIONSEQUENCE (SEQ ID NO:) huOKT3 V_(L)DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 16) huOKT3 V_(L); V_(L)-DIQMNTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKR Q100CWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWS SNPFTFG

GTKLQITR (SEQ ID NO: 65)

TABLE 6 scFv Sequence.The uppercase, non-italicized, non-bold, non-underlined sequence represents the V_(H)domain, The uppercase, italicized sequence represents the V_(L) domain. The uppercase,underlined, italicized, and bold sequences represent the mutations described in the″DESCRIPTION″ column, The lowercase bold sequences represent the intra-scFv linker.DESCRIPTION SEQUENCE (SEQ ID NO:) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S, 15 aminoGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP acid intra-scFvEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggs DI linkerQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 19) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; 5 amino acidGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP intra-scFv linkerEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggs DIQMTQSPSS LSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 48)huOKT3 scFv QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; 10 aminoGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP acid intra-scFvEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggs DIQMT linkerQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKL QITR (SEQ ID NO: 49)huOKT3 scFv QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; 20 aminoGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP acid intra-scFvEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg linker ggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 50) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; 25 aminoGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP acid intra-scFvEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg linker ggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 51) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; 30 aminoGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP acid intra-scFvEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg linker ggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQIRT (SEQ ID NO: 52) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

CLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 5 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggs DIQMTQSPSS acid intra-scFvLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPS linkerRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFG

GTKLQITR (SEQ ID NO: 53) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

LEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 10 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSS ggggsggggs DIQMT acid intra-scFvQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLA linkerSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFG

GTKL QITR (SEQ ID NO: 54) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

LEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 15 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggs DI acid intra-scFvQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDT linkerSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFG

GTKLQTIR (SEQ ID NO: 55) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

LEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 20 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg acid intra-scFvggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRW linkerIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFT FG

GTKLQITR (SEQ ID NO. 56) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

LEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 25 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg acid intra-scFv ggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKA linkerPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWS SNPFTFG

GTKLQITR (SEQ ID NO: 57) huOKT3 scFvQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK C105S; VL-Q100C;

LEWIGYINPSRGYTNYNQKEKDRFTISRDNSKNTAFLQMDSLRP VH-G44C; 30 aminoEDTGVYFCARYYDDHY

LDYWGQGTPVTVSSggggsggggsggggsgg acid intra-scFv ggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTP linkerGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQ QWSSNPFTFG

GTKLQITR (SEQ ID NO: 58) huOKT3; 15 aminoQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK acid intra-scEvGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRP linkerEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSggggsggggsggggs DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 59)

TABLE 7 Light Chain Fusion Polypeptide Sequence.The uppercase, non-italicized, non-bold, non-underlined sequence represents the V_(L) domainof the trastuzumab light chain. The uppercase, italicized sequence represents the constantregion of the trastuzumab light chain. The lowercase, non-italicized, non-bold, non-underlinedsequence represents the linker conjugating the light chain to the scFv. The uppercase,underlined sequence represents the V_(H) domain of the scFv, The uppercase, bold sequencerepresents the V_(L) domain of the scFv, The uppercase, underlined, italicized, and bold sequencesrepresent the mutations described in the ″DESCRIPTION″ column, The lowercase bold sequencesrepresent the intra-scFv linker. DESCRIPTION SEQUENCE (SEQ ID NO:)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S; 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the scFv; 15KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFv peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 29) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; C105S, 17KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH amino acid linkerYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF conjugating the lightYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE chain to the scFv, 5KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFV peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTK LQITR (SEQ ID NO: 30)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the scFv, 10KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFV peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFG QGTKLQITR (SEQ ID NO: 31)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the scFv, 20KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFV peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 32) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; C105S, 17KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH amino acid linkerYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF conjugating the lightYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE chain to the scFv, 25KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFV peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 33) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; C105S, 17KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH amino acid linkerYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF conjugating the lightYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE chain to the scFv, 30KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS amino acid intra-GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGY scFV peptide linkerINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 34) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; C105S, 17KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH amino acid linkerYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF conjugating the lightYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE chain to the huOKT3KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS scFv; 5 amino acidGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsDIQMTQSPSSLSASVG V_(H)-G44CDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTK LQITR (SEQ ID NO: 42)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the huOKT3 KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQSscFv; 10 amino acid GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsDIQMTQSPSSLS V_(H)-G44CASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFG CGTKLQITR (SEQ ID NO: 43)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the huOKT3 KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQSscFv; 15 amino acid GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsgggsDIQMTQSP V_(H)-G44CSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPF TFGCGTKLQITR (SEQ ID NO: 44)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the huOKT3 KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQSscFv; 20 amino acid GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsgggsggggsDIQMT V_(H)-G44CQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSS NPFTFGCGTKLQITR (SEQ ID NO: 45)Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPchain; C105S, 17 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHamino acid linker YTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFconjugating the light YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEchain to the huOKT3 KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQSscFv; 25 amino acid GGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsgggsggggsggggsDI V_(H)-G44CQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR (SEQ ID NO: 46) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; C105S, 17KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH amino acid linkerYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF conjugating the lightYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE chain to the huOKT3KHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS scFv; 30 amino acidGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK

LEWIGYI intra-scFv peptide NPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFlinker; V_(L)-Q100C; CARYYDDHY

LDYWGQGTPVTVSS ggggsggggsgggsggggsggggsgg V_(H)-G44CggsDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR (SEQ ID NO: 47) Trastuzumab lightDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP chain; 17 amino acidKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH linker conjugatingYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF the light chain to theYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE scFv; 15 amino acidKHKVYACEVTHQGLSSPVTKSFNRGECtsggggsggggsggggsQVQLVQS intra-scFv peptideGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYI linkerNPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF CARYYDDHYCLDYWGQGTPVTVSSggggsggggsgggsDIQMTQSP SSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPF TFGCGTKLQITR (SEQ ID NO: 47)

TABLE 8 Modifications to bispecific binding molecules LOCA- TION OFMODIFI- CATION DESCRIPTION Heavy chain Mutation to reduce binding to theFc receptor (as an example, N297A mutation) Mutation to destroy aglycosylation site (as an example, N297A mutation) Mutation to reduceClq binding (as an example, K322A mutation) Linker Increase or decreasethe length of the linker conjugating the light chain to the huOKT3 scFvhuOKT3 Mutation to increase stabilization and/or reduce scFv V_(H)aggregation (as an example, introduce disulfide binding between V_(H)40and V_(L)100 (according to Kabat numbering), as an example, V_(H) G44Cand V_(L) Q100C) Reduce aggregation (as an example, C105S mutation)huOKT3 Mutation to increase stabilization and/or reduce scFv V_(L)aggregation (as an example,, introduce disulfide binding between V_(H)40and V_(L)100 (according to Kabat numbering), as an example, V_(H) G44Cand V_(L) Q100C) huOKT3 Increase or decrease the length of the linkerintra-scFv linker

5.2 Bispecific Binding Molecule Conjugates

In preferred embodiments, a bispecific binding molecule provided hereinis not conjugated to any other molecule, such as an organic moiety, adetectable label, or an isotope. In alternative embodiments, abispecific binding molecule provided herein is conjugated to one or moreorganic moieties. In alternative embodiments, a bispecific bindingmolecule provided herein is conjugated to one or more detectable labels.In alternative embodiments, a bispecific binding molecule providedherein is conjugated to one or more isotopes.

5.2.1 Detectable Labels and Isotopes

In certain embodiments, a bispecific binding molecule provided herein isconjugated to one or more detectable labels or isotopes, e.g., forimaging purposes. In certain embodiments, a bispecific binding moleculeis detectably labeled by covalent or non-covalent attachment of achromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic,chemiluminescent, nuclear magnetic resonance contrast agent or otherlabel.

Non-limiting examples of suitable chromogenic labels includediaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid.

Non-limiting examples of suitable enzyme labels include malatedehydrogenase, staphylococcal nuclease, delta-S-steroid isomerase,yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase,triose phosphate isomerase, peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase, andacetylcholine esterase.

Non-limiting examples of suitable radioisotopic labels include ³H,¹¹¹In, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu,⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ²²³Ra, ²²³Ra, ⁸⁹Zr, ¹⁷⁷Lu, and¹⁰⁹Pd. In certain embodiments, ¹¹¹In is a preferred isotope for in vivoimaging as it avoids the problem of dehalogenation of ¹²⁵I or¹³¹I-labeled bispecific binding molecules in the liver. In addition,¹¹¹In has a more favorable gamma emission energy for imaging (Perkins etal., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et ah, J. Nucl.Med. 25:281-287 (1987)). For example, ¹¹¹In coupled to monoclonalantibodies with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptakein non-tumorous tissues, particularly the liver, and therefore enhancesspecificity of tumor localization (Esteban et al., J. Nucl. Med.28:861-870 (1987)).

Non-limiting examples of suitable non-radioactive isotopic labelsinclude ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Non-limiting examples of suitable fluorescent labels include a ¹⁵²Eulabel, a fluorescein label, an isothiocyanate label, a rhodamine label,a phycoerythrin label, a phycocyanin label, an allophycocyanin label, aGreen Fluorescent Protein (GFP) label, an o-phthaldehyde label, and afluorescamine label.

Non-limiting examples of chemiluminescent labels include a luminollabel, an isoluminol label, an aromatic acridinium ester label, animidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, and an aequorin label.

Non-limiting examples of nuclear magnetic resonance contrasting agentsinclude heavy metal nuclei such as Gd, Mn, and iron.

Techniques known to one of ordinary skill in the art for binding theabove-described labels to a bispecific binding molecule provided hereinare described in, for example, Kennedy et at., Clin. CMm. Acta 70:1-31(1976), and Schurs et al., Clin. CMm. Acta 81:1-40 (1977). Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

In certain embodiments, the bispecific binding molecule is conjugated toa diagnostic agent. A diagnostic agent is an agent useful in diagnosingor detecting a disease by locating the cells containing the antigen.Useful diagnostic agents include, but are not limited to, radioisotopes,dyes (such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.,paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No.6,331,175 describes MRI technique and the preparation of antibodiesconjugated to a MRI enhancing agent and is incorporated in its entiretyby reference. Preferably, the diagnostic agents are selected from thegroup consisting of radioisotopes, enhancing agents for use in magneticresonance imaging, and fluorescent compounds. In order to load anantibody component with radioactive metals or paramagnetic ions, it maybe necessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which can bebound chelating groups such as, for example, ethylenediaminetetraaceticacid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and likegroups known to be useful for this purpose. Chelates are coupled to theantibodies using standard chemistries. The chelate is normally linked tothe antibody by a group which enables formation of a bond to themolecule with minimal loss of immunoreactivity and minimal aggregationand/or internal cross-linking other, more unusual, methods and reagentsfor conjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates,” issued Apr. 25,1989, the disclosure of which is incorporated herein in its entirety byreference. Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes for radio-imaging. The same chelates, when complexedwith non-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along bispecific binding molecules providedherein. Macrocyclic chelates such as NOTA, DOTA, and TETA are of usewith a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT are encompassed herein.

5.2.2 Organic Conjugates

In certain embodiments, the bispecific binding molecules provided hereincomprise one or more organic moieties that are covalently bonded,directly or indirectly, to the bispecific binding molecule. Suchmodification can produce an antibody or antigen-binding fragment withimproved pharmacokinetic properties (e.g., increased in vivo serumhalf-life). The organic moiety can be a hydrophilic polymeric group,fatty acid group, or fatty acid ester group. As used herein, the term“fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids.As used herein, a “hydrophilic polymeric group” refers to an organicpolymer that is more soluble in water than in octane, e.g., polylysine.Hydrophilic polymers suitable for modifying a bispecific bindingmolecule provided herein can be linear or branched and include, forexample, polyalkane glycols (e.g., polyethylene glycol, (PEG),monomethoxy-polyethylene glycol, and polypropylene glycol),carbohydrates (e.g., dextran, cellulose, oligosaccharides, andpolysaccharides), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, and polyaspartate), polyalkane oxides (e.g., polyethyleneoxide and polypropylene oxide) and polyvinyl pyrolidone. In certainembodiments, the hydrophilic polymer that modifies a bispecific bindingmolecule provided herein has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying bispecificbinding molecules provided herein can be saturated or can contain one ormore units of unsaturation. Fatty acids that are suitable for modifyingbispecific binding molecules provided herein include, for example,n-dodecanoate, n-tetradecanoate, n-octadecanoate, n-eicosanoate,n-docosanoate, n-triacontanoate, n-tetracontanoate,cis-delta-9-octadecanoate, all cis-delta-5,8,11,14-eicosatetraenoate,octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The bispecific binding molecule conjugates provided herein can beprepared using suitable methods, such as by reaction with one or moremodifying agents. As used herein, an “activating group” is a chemicalmoiety or functional group that can, under appropriate conditions, reactwith a second chemical group thereby forming a covalent bond between themodifying agent and the second chemical group. For example,amine-reactive activating groups include electrophilic groups such as,for example, tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups thatcan react with thiols include, for example, maleimide, iodoacetyl,acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol(TNB-thiol), and the like. An aldehyde functional group can be coupledto amine- or hydrazide-containing molecules, and an azide group canreact with a trivalent phosphorous group to form phosphoramidate orphosphorimide linkages. Suitable methods to introduce activating groupsinto molecules are known in the art (see, for example, Hernanson, G. T.,Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Anactivating group can be bonded directly to the organic group (e.g.,hydrophilic polymer, fatty acid, fatty acid ester), or through a linkermoiety, for example a divalent C₁-C₁₂ group, wherein one or more carbonatoms can be replaced by a heteroatom such as oxygen, nitrogen orsulfur. Suitable linker moieties include, for example, tetraethyleneglycol, (CH₂)₃, and NH. Modifying agents that comprise a linker moietycan be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine or mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

As used herein, a “modifying agent” refers to a suitable organic group(e.g., hydrophilic polymer, a fatty acid, and a fatty acid ester) thatcomprises an activating group. For example, the organic moieties can bebonded to the bispecific binding molecule in a non-site specific mannerby employing an amine-reactive modifying agent, for example, anN-hydroxysuccinimide ester of PEG. Modified bispecific binding moleculescan also be prepared by reducing disulfide bonds (e.g., intra-chaindisulfide bonds) of bispecific binding molecule. The reduced bispecificbinding molecule can then be reacted with a thiol-reactive modifyingagent to produce the modified bispecific binding molecule providedherein. Modified bispecific binding molecules comprising an organicmoiety that is bonded to specific sites of a bispecific binding moleculeprovided herein can be prepared using suitable methods, such as reverseproteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlenet al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., ProteinSci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68(1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), andthe methods described in Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996).

5.3 Bispecific Binding Molecule Production

Provided herein are methods for producing bispecific binding moleculesas described in Section 5.1 and Section 5.2. In certain embodiments,provided herein are methods for producing a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, comprising two identical heavy chainsand two identical light chains, said light chains being a first lightchain and a second light chain, wherein the first light chain is fusedto a first single chain variable fragment (scFV), via a peptide linker,to create a first fusion polypeptide, and wherein the second light chainis fused to a second scFv, via a peptide linker, to create a secondfusion polypeptide, wherein the first and second scFv (i) are identical,and (ii) bind to CD3, and wherein the first and second fusionpolypeptides are identical.

Methods to produce bispecific binding molecules described herein areknown to one of ordinary skill in the art, for example, by chemicalsynthesis, by purification from biological sources, or by recombinantexpression techniques, including, for example, from mammalian cell ortransgenic preparations. The methods described herein employs, unlessotherwise indicated, conventional techniques in molecular biology,microbiology, genetic analysis, recombinant DNA, organic chemistry,biochemistry, PCR, oligonucleotide synthesis and modification, nucleicacid hybridization, and related fields within the skill of the art.These techniques are described, for example, in the references citedherein and are fully explained in the literature. See, for example,Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press; Sambrook et al. (1989), MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press; Sambrook et al. (2001) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons (1987 and annual updates); Current Protocols in Immunology, JohnWiley & Sons (1987 and annual updates) Gait (ed.) (1984) OligonucleotideSynthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991)Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birrenet al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold SpringHarbor Laboratory Press.

A variety of methods exist in the art for the production of bispecificbinding molecules. For example, the bispecific binding molecule may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. The one or more DNAs encoding a bispecific bindingmolecule provided herein can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of murine antibodies, or such chains from human, humanized, orother sources). Once isolated, the DNA may be placed into expressionvectors, which are then transformed into host cells such as NSO cells,Simian COS cells, Chinese hamster ovary (CHO) cells, yeast cells, algaecells, eggs, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the bispecificbinding molecules in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains of a desired species in place ofthe homologous human sequences (U.S. Pat. No. 4,816,567; Morrison etal., supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of a bispecific binding molecule providedherein. In certain embodiments, the DNA is as described in Section5.3.1.

Bispecific binding molecules provided herein can also be prepared usingat least one bispecific binding molecule-encoding polynucleotide toprovide transgenic animals or mammals, such as goats, cows, horses,sheep, and the like, that produce such antibodies in their milk. Suchanimals can be provided using known methods. See, for example, but notlimited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616, 5,565,362; 5,304,489, and the like, each of which is entirelyincorporated herein by reference.

In certain embodiments, bispecific binding molecules provided herein canadditionally be prepared using at least one bispecific bindingmolecule-encoding polynucleotide provided herein to provide transgenicplants and cultured plant cells (for example, but not limited to tobaccoand maize) that produce such antibodies, specified portions or variantsin the plant parts or in cells cultured therefrom. As a non-limitingexample, transgenic tobacco leaves expressing recombinant proteins havebeen successfully used to provide large amounts of recombinant proteins,for example, using an inducible promoter. See, for example, Cramer etal., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and referencescited therein. Also, transgenic maize have been used to expressmammalian proteins at commercial production levels, with biologicalactivities equivalent to those produced in other recombinant systems orpurified from natural sources. See, for example, Hood et al., Adv. Exp.Med. Biol. 464:127-147 (1999) and references cited therein. Antibodieshave also been produced in large amounts from transgenic plant seedsincluding antibody fragments, such as scFvs, including tobacco seeds andpotato tubers. See, for example, Conrad et al., Plant Mol. Biol.38:101-109 (1998) and references cited therein. Thus, bispecific bindingmolecules can also be produced using transgenic plants, according toknown methods. See also, for example, Fischer et al., Biotechnol. Appl.Biochem. 30:99-108 (October, 1999), Ma et al., Trends Biotechnol.13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam etal., Biochem Soc. Trans. 22:940-944 (1994); and references citedtherein. Each of the above references is entirely incorporated herein byreference.

In certain embodiments, bispecific binding molecules provided herein canbe prepared using at least one bispecific binding molecule-encodingpolynucleotide provided herein to provide bacteria that produce suchbispecific binding molecules. As a non-limiting example, E. coliexpressing recombinant proteins has been successfully used to providelarge amounts of recombinant proteins. See, for example, Verma et al.,1998, 216(1-2): 165-181 and references cited therein.

See, also, Section 6.1.2.1 for a detailed example for the design andproduction of a bispecific binding molecule described herein.

In certain embodiments, the bispecific binding molecules can berecovered and purified from recombinant cell cultures by well-knownmethods including, but not limited to, protein A purification, protein Gpurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, for example, Colligan, CurrentProtocols in Immunology, or Current Protocols in Protein Science, JohnWiley & Sons, NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and10, each entirely incorporated herein by reference.

In certain embodiments, the bispecific binding molecules provided hereininclude naturally purified products, products of chemical syntheticprocedures, and products produced by recombinant techniques from aeukaryotic host, including, for example, yeast, higher plant, insect andmammalian cells. In preferred embodiments, the bispecific bindingmolecule is generated in a host such that the bispecific bindingmolecule is aglycosylated. In another preferred embodiment, thebispecific binding molecule is generated in a bacterial cell such thatthe bispecific binding molecule is aglycosylated. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and20, Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

Purified antibodies can be characterized by, for example, ELISA,ELISPOT, flow cytometry, immunocytology, Biacore™ analysis, SapidyneKinExA™ kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLCanalysis as well as by a number of other functional assays disclosedherein.

5.3.1 Polynucleotides

In certain embodiments, provided herein are polynucleotides comprising anucleotide sequence encoding a bispecific binding molecule describedherein or a fragment thereof (e.g., a heavy chain and/or a light chainfusion polypeptide) that immunospecifically binds to HER2 and CD3, asdescribed in Section 5.1 and Section 5.2. Also provided herein arevectors comprising such polynucleotides. See, Section 5.3.2. Alsoprovided herein are polynucleotides encoding antigens of the bispecificbinding molecules provided herein. Also provided herein arepolynucleotides that hybridize under stringent or lower stringencyhybridization conditions to polynucleotides that encode a bispecificbinding molecule or fragment thereof provided herein.

The language “purified” includes preparations of polynucleotide ornucleic acid molecule having less than about 15%, 10%, 5%, 2%, 1%, 0.5%,or 0.1% (in particular less than about 10%) of other material, e.g.,cellular material, culture medium, other nucleic acid molecules,chemical precursors and/or other chemicals. In a specific embodiment, anucleic acid molecule(s) encoding a bispecific binding moleculedescribed herein is isolated or purified.

Nucleic acid molecules provided herein can be in the form of RNA, suchas mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

In certain embodiments, provided herein is a polynucleotide comprisingnucleotide sequences encoding a bispecific binding molecule or fragmentthereof as described in Section 5.1 and Section 5.2, wherein thebispecific binding molecule comprises an aglycosylated monoclonalantibody that is an immunoglobulin that binds to HER2, comprising twoidentical heavy chains and two identical light chains, said light chainsbeing a first light chain and a second light chain, wherein the firstlight chain is fused to a first scFv, via a peptide linker, to create afirst light chain fusion polypeptide, and wherein the second light chainis fused to a second scFv, via a peptide linker, to create a secondlight chain fusion polypeptide, wherein the first and second scFv (i)are identical, and (ii) bind to CD3, and wherein the first and secondlight chain fusion polypeptides are identical.

For a detailed example for the generation of a bispecific bindingmolecule as described herein, see, Section 6.1.2.1 for a detailedexample for the design and production of a bispecific binding moleculedescribed herein.

In certain embodiments, provided herein is a polynucleotide comprisingnucleotide sequences encoding a light chain fusion polypeptidecomprising a light chain fused to a scFv, via a peptide linker, whereinthe light chain binds to HER2 and wherein the scFv binds to CD3. Incertain embodiments, the light chain is the light chain of aHER2-specific antibody known in the art, such as, for example,trastuzumab, M-111, pertuzumab, ertumaxomab, MDXH210, 2B1, and MM-302.In certain embodiments, the scFv comprises the V_(H) and V_(L) of ananti-CD3 antibody known in the art, such as, for example, huOKT3,YTH12.5, HUM291, teplizumab, huCLB-T3/4, otelixizumab, blinatumomab, MTI10, catumaxomab, 28F11, 27H5, 23F10, 15C3, visilizumab, and Hum291. In apreferred embodiment, the anti-CD3 antibody is huOKT3. In an especiallypreferred embodiment, the scFv comprises the V_(H) of huOKT3, furthercomprising the amino acid substitution at numbered position 105, whereinthe cysteine is substituted with a serine. See, for example, Kipriyanovet al. 1997, Protein Eng. 445-453. In certain embodiments, the scFv isderived from the huOKT3 monoclonal antibody and comprises one or moremutations, relative to the native huOKT3 V_(H) and V_(L), to stabilizedisulfide binding. In certain embodiments, the stabilization ofdisulfide binding prevents aggregation of the bispecific bindingmolecule. In certain embodiments, the stabilization of disulfide bindingreduces aggregation of the bispecific binding molecule as compared toaggregation of the bispecific binding molecule without the stabilizationof disulfide binding. In certain embodiments of the bispecific bindingmolecule, the one or more mutations to stabilize disulfide bindingcomprise a V_(H) G44C mutation and a V_(L) Q100C mutation (e.g., aspresent in SEQ ID NOS: 54-59). In certain embodiments of the bispecificbinding molecule, the one or more mutations to stabilize disulfidebinding are the replacement of the amino acid residue at V_(H)44(according to the Kabat numbering system) with a cysteine and thereplacement of the amino acid residue at V_(L)100 (according to theKabat numbering system) with a cysteine so as to introduce a disulfidebond between V_(H)44 and V_(L)100 (e.g., as present in SEQ ID NOS:54-59). In certain embodiments, the peptide linker is between 5-30,5-25, 5-15, 10-30, 10-20, 10-15, 15-30, or 15-25 amino acid residues inlength. In certain embodiments, the sequence of the peptide linker is asdescribed in Table 1, above (e.g., any one of SEQ ID NOs: 14 or 35-41).In a particularly preferred embodiment, the sequence of the peptidelinker is SEQ ID NO: 14. In certain embodiments, the sequence to thescFv comprises one or more modifications as described in Table 8, above.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding bispecific binding molecules or fragmentsthereof, which specifically bind to HER2 and CD3, and comprise an aminoacid sequence as described herein, as well as antibodies which competewith such bispecific binding molecules for binding to HER2 and/or CD3,or which binds to the same epitope as that of such antibodies.

In a preferred embodiment, the sequence of the light chain is SEQ ID NO:25. In a preferred embodiment, the nucleotide sequence encoding thelight chain is SEQ ID NO: 24. In a preferred embodiment, the sequence ofthe scFv SEQ ID NO: 19. In a preferred embodiment, the nucleotidesequence encoding the scFv SEQ ID NO: 18. In a preferred embodiment, thesequence of the light chain is SEQ ID NO: 25 and the sequence of thescFv is SEQ ID NO: 19. In a preferred embodiment, the nucleotidesequence encoding the light chain is SEQ ID NO: 24 and the nucleotidesequence encoding the scFv is SEQ ID NO: 18. In a preferred embodiment,the sequence of the light chain fusion polypeptide is SEQ ID NO: 29. Ina preferred embodiment, the nucleotide sequence encoding the light chainfusion polypeptide is SEQ ID NO: 28.

In certain embodiments, the bispecific binding molecule has atrastuzumab-derived sequence that contains one or more of themodifications in the trastuzumab immunoglobulin, and has ahuOKT3-derived sequence that contains one or more of the modificationsin the huOKT3 V_(H) and V_(L) sequences, as described in Table 8, below.Bispecific binding molecules having other immunoglobulin or scFvsequences can contain analogous mutations at corresponding positions inthese other immunoglobulin or scFv sequences. In certain embodiments,the bispecific binding molecule is (a) derived from trastuzumab andhuOKT3; and (b) contains one or more of the modifications as describedin Table 8, above. In certain embodiments, the sequence of the peptidelinker conjugating the immunoglobulin light chain and the scFv is asdescribed in Table 1, above (e.g., any one of SEQ ID NOs: 14 or 35-41).In certain embodiments, the sequence of the heavy chain is as describedin Table 2, above (e.g., any one of SEQ ID NOs: 23, 27, 62, or 63). Incertain embodiments, the sequence of the light chain is as described inTable 3, above (e.g., SEQ ID NO: 25). In certain embodiments, thesequence of the V_(H) of the scFv is as described in Table 4, above(e.g., any one of SEQ ID NOs: 15, 17, or 64). In certain embodiments,the sequence of the V_(L) of the scFv is as described in Table 5, above(e.g., any one of SEQ ID NOs: 16 or 65). In certain embodiments, thesequence of the scFv peptide linker is as described in Table 1, above(e.g., any one of SEQ ID NOs: 14 or 35-41). In certain embodiments, thesequence of the scFv is as described in Table 6, above (e.g., any one ofSEQ ID NOs: 19 or 48-59). In certain embodiments, the sequence of thelight chain fusion polypeptide is as described in Table 7, above (e.g.,any one of SEQ ID NOs: 29, 34, 42-47, or 60).

In certain embodiments, provided herein is a polynucleotide comprisingnucleotide sequences encoding the heavy chain of a HER2-specificantibody described in Section 5.2. In certain embodiments, the heavychain is the heavy chain a HER2-specific antibody known in the art, suchas, for example, trastuzumab, M-111, pertuzumab, ertumaxomab, MDXH210,2B1, and MM-302. In a preferred embodiment, the antibody comprises theV_(H) of trastuzumab, wherein the sequence of the heavy chain is SEQ IDNO: 27. In a preferred embodiment, the antibody comprises the V_(H) oftrastuzumab, wherein the nucleotide sequence encoding the heavy chain isSEQ ID NO: 26. In a preferred embodiment, the sequence of the heavychain is comprises the V_(H) of trastuzumab and comprises the amino acidsubstitution N297A in the Fc region (SEQ ID NO: 26). In a preferredembodiment, the nucleotide sequence encoding the heavy chain comprisesthe nucleotide sequence encoding the trastuzumab V_(H) and comprises theamino acid substitution N297A in the Fc region (SEQ ID NO: 26). Inpreferred embodiment, the sequence of the heavy chain comprises thesequence of the trastuzumab V_(H) and comprises the amino acidsubstitution K322A in the Fc region (SEQ ID NO: 27). In a preferredembodiment, the nucleotide sequence encoding the heavy chain comprisesthe nucleotide sequence encoding the trastuzumab V_(H) and comprises theamino acid substitution K322A in the Fc region (SEQ ID NO: 26). In anespecially preferred embodiment, the sequence of the heavy chaincomprises the sequence of the trastuzumab V_(H) and comprises the aminoacid substitutions N297A and K322A in the Fc region (SEQ ID NO: 27). Inan especially preferred embodiment, the nucleotide sequence encoding theheavy chain comprises the nucleotide sequence encoding the trastuzumabV_(H) and comprises the amino acid substitutions N297A and K322A in theFc region (SEQ ID NO: 26).

The polynucleotides provided herein can be obtained by any method knownin the art. For example, if the nucleotide sequence encoding abispecific binding molecule or fragment thereof described herein isknown, a polynucleotide encoding the bispecific binding molecule orfragment thereof can be may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding a bispecific binding moleculeor fragment thereof may be generated from nucleic acid from a suitablesource. If a clone containing a nucleic acid encoding a particularbispecific binding molecule or fragment thereof is not available, butthe sequence of the bispecific binding molecule or fragment thereof isknown, a nucleic acid encoding the bispecific binding molecule orfragment thereof may be chemically synthesized or obtained from asuitable source (e.g., an antibody cDNA library, or a cDNA librarygenerated from, or nucleic acid, preferably poly A+RNA, isolated from,any tissue or cells expressing the antibody, such as hybridoma cellsselected to express an antibody provided herein) by PCR amplificationusing synthetic primers that hybridize to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, for example, a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art. See, for example, Section 5.3.2.

In certain embodiments, the amino acid sequence of the antibody of thebispecific binding molecule is known in the art. In such embodiments, apolypeptide encoding such an antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., whichare both incorporated by reference herein in their entireties), togenerate bispecific binding molecules having a different amino acidsequence, for example, to create amino acid substitutions, deletions,and/or insertions. For example, such manipulations can be performed torender the encoded amino acid aglycosylated, or to destroy theantibody's ability to bind to C1q, Fc receptor, or to activate thecomplement system.

Isolated nucleic acid molecules provided herein can include nucleic acidmolecules comprising an open reading frame (ORF), optionally with one ormore introns, for example, but not limited to, at least one specifiedportion of at least one complementarity determining region (CDR), asCDR1, CDR2 and/or CDR3 of at least one heavy chain or light chain;nucleic acid molecules comprising the coding sequence for an anti-HER2antibody or variable region, an anti-CD3 scFv, or a single chain fusionpolypeptide; and nucleic acid molecules which comprise a nucleotidesequence substantially different from those described above but which,due to the degeneracy of the genetic code, still encode at least onebispecific binding molecule as described herein and/or as known in theart.

Also provided herein are isolated nucleic acids that hybridize underselective hybridization conditions to a polynucleotide disclosed herein.Thus, the polynucleotides of this embodiment can be used for isolating,detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides provided herein can beused to identify, isolate, or amplify partial or full-length clones in adeposited library. In some embodiments, the polynucleotides are genomicor cDNA sequences isolated, or otherwise complementary to, a cDNA from ahuman or mammalian nucleic acid library.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide provided herein. For example, a multi-cloning sitecomprising one or more endonuclease restriction sites can be insertedinto the nucleic acid to aid in isolation of the polynucleotide. Inaddition, translatable sequences can be inserted to aid in the isolationof the translated polynucleotide provided herein. For example, ahexa-histidine marker sequence provides a convenient means to purify thepolypeptides provided herein. The nucleic acid provided herein—excludingthe coding sequence—is optionally a vector, adapter, or linker forcloning and/or expression of a polynucleotide provided herein.

Additional sequences can also be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

In a specific embodiment, using routine recombinant DNA techniques, oneor more of the CDRs of an antibody described herein may be insertedwithin framework regions. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds HER2. One or more aminoacid substitutions may be made within the framework regions, and,preferably, the amino acid substitutions improve binding of the antibodyto its antigen. Additionally, such methods may be used to make aminoacid substitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are provided herein and within theskill of the art.

In certain embodiments, the isolated or purified nucleic acid molecule,or fragment thereof, upon linkage with another nucleic acid molecule,can encode a fusion protein. The generation of fusion proteins is withinthe ordinary skill in the art and can involve the use of restrictionenzyme or recombinational cloning techniques (see, for example,Gateway™. (Invitrogen)). See, also, U.S. Pat. No. 5,314,995.

In certain embodiments, a polynucleotide provided herein is in the formof a vector (e.g., expression vector) as described in Section 5.3.2.

5.3.2 Cells and Vectors

In certain embodiments, provided herein are cells (e.g., ex vivo cells)expressing (e.g., recombinantly) bispecific binding molecules asdescribed herein. Also provided herein are vectors (e.g., expressionvectors) comprising nucleotide sequences (see, for example, Section5.3.1) encoding a bispecific binding molecule or fragment thereofdescribed herein for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are cells (e.g., ex vivo cells)comprising such vectors or nucleotide sequences for recombinantlyexpressing a bispecific binding molecule described here. Also providedherein are methods for producing a bispecific binding molecule describedherein, comprising expressing such bispecific binding molecule from acell (e.g., ex vivo cell). In a preferred embodiment, the cell is an exvivo cell.

A vector (e.g., expression vector) is a DNA molecule comprising a genethat is expressed in a cell (e.g., ex vivo cell). Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements, e.g., a promoter. A recombinant hostmay be any prokaryotic or eukaryotic cell that contains either a cloningvector or expression vector. This term also includes those prokaryoticor eukaryotic cells, as well as a transgenic animal, that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell or cells of the host cells (e.g., ex vivocells).

In a preferred embodiment, the promoter is the CMV promoter.

In certain embodiments, provided herein is a vector comprising one ormore polynucleotide as described in Section 5.3.1.

In certain embodiments, a polynucleotide as described in Section 5.3.1can be cloned into a suitable vector and can be used to transform ortransfect any suitable host. Vectors and methods to construct suchvectors are known to one of ordinary skill in the art and are describedin general technical references (see, in general, “Recombinant DNA PartD,” Methods in Enzymology, Vol. 153, Wu and Grossman, eds., AcademicPress (1987)). In certain embodiments, the vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, insect, or mammal) into which the vector is tobe introduced, as appropriate and taking into consideration whether thevector is DNA or RNA. In certain embodiments, the vector comprisesregulatory sequences that are specific to the genus of the host. Incertain embodiments, the vector comprises regulatory sequences that arespecific to the species of the host.

In certain embodiments, the vector comprises one or more marker genes,which allow for selection of transformed or transfected hosts.Non-limiting examples of marker genes include biocide resistance, e.g.,resistance to antibiotics, heavy metals, etc., complementation in anauxotrophic host to provide prototrophy, and the like. In a preferredembodiment, the vector comprises ampicillin and hygromycin selectablemarkers.

In certain embodiments, an expression vector can comprise a native ornormative promoter operably linked to a polynucleotide as described inSection 5.3.1. The selection of promoters, for example, strong, weak,inducible, tissue-specific and developmental-specific, is within theskill in the art. Similarly, the combining of a nucleic acid molecule,or fragment thereof, as described above with a promoter is also withinthe skill in the art.

Non-limiting examples of suitable vectors include those designed forpropagation and expansion or for expression or both. For example, acloning vector can be selected from the group consisting of the pUCseries, the pBluescript series (Stratagene, LaJolla, Calif.), the pETseries (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).Bacteriophage vectors, such as lamda-GT10, lamda-GT11, lamda-ZapII(Stratagene), lamda-EMBL4, and lamda-NM1149, can also be used.Non-limiting examples of plant expression vectors include pBI110,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Non-limiting examplesof animal expression vectors include pEUK-C1, pMAM and pMAMneo(Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) canalso be used in accordance with the manufacturer's recommendations.

In certain embodiments, the vector is a mammalian vector. In certainembodiments, the mammalian vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, thebispecific binding molecule coding sequence, and signals required forthe termination of transcription and polyadenylation of the transcript.In certain embodiments, the mammalian vector contains additionalelements, such as, for example, enhancers, Kozak sequences andintervening sequences flanked by donor and acceptor sites for RNAsplicing. In certain embodiments, highly efficient transcription can beachieved with, for example, the early and late promoters from SV40, thelong terminal repeats (LTRS) from retroviruses, for example, RSV, HTLVI,HIVI and the early promoter of the cytomegalovirus (CMV). However,cellular elements can also be used (e.g., the human actin promoter).Non-limiting examples of mammalian expression vectors include, vectorssuch as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (ClonetechLabs, Palo Alto, Calif.), pcDNA3.1 (+/−), pcDNA/Zeo (+/−) orpcDNA3.1/Hygro (+/−)(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBCI2MI (ATCC67109). Non-limiting example of mammalian host cells that can be used incombination with such mammalian vectors include human Hela 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

In certain embodiments, the vector is a viral vector, for example,retroviral vectors, parvovirus-based vectors, e.g., adeno-associatedvirus (AAV)-based vectors, AAV-adenoviral chimeric vectors, andadenovirus-based vectors, and lentiviral vectors, such as Herpes simplex(HSV)-based vectors. In certain embodiments, the viral vector ismanipulated to render the virus replication deficient. In certainembodiments, the viral vector is manipulated to eliminate toxicity tothe host. These viral vectors can be prepared using standard recombinantDNA techniques described in, for example, Sambrook et al., MolecularCloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons,New York, N.Y. (1994).

In certain embodiments, a vector or polynucleotide described herein canbe transferred to a cell (e.g., an ex vivo cell) by conventionaltechniques and the resulting cell can be cultured by conventionaltechniques to produce a bispecific binding molecule described herein.Accordingly, provided herein are cells comprising a polynucleotideencoding a bispecific binding molecule or fragment thereof, a heavy orlight chain thereof, or a light chain fusion polypeptide thereof,operably linked to a promoter for expression of such sequences in thehost cell. In certain embodiments, a vector encoding the heavy chainoperably linked to a promoter and a vector encoding the light chainfusion polypeptide operably linked to a promoter can be co-expressed inthe cell for expression of the entire bispecific binding molecule, asdescribed below. In certain embodiments, a cell comprises a vectorcomprising a polynucleotide encoding both the heavy chain and the lightchain fusion polypeptide of a bispecific binding molecule describedherein operably linked to a promoter. In certain embodiments, a cellcomprises two different vectors, a first vector comprising apolynucleotide encoding a heavy chain operably linked to a promoter, anda second vector comprising a polynucleotide encoding a light chainfusion polypeptide operably linked to a promoter. In certainembodiments, a first cell comprises a first vector comprising apolynucleotide encoding a heavy chain of a bispecific binding moleculedescribed herein, and a second cell comprises a second vector comprisinga polynucleotide encoding a light chain fusion polypeptide of abispecific binding molecule described herein. In certain embodiments,provided herein is a mixture of cells comprising such first cell andsuch second cell. In a preferred embodiment, the cell expresses thevector or vectors such that the oligonucleotide is both transcribed andtranslated efficiently by the cell.

In embodiment, the cell expresses the vector, such that theoligonucleotide, or fragment thereof, is both transcribed and translatedefficiently by the cell.

In certain embodiments, the cell is present in a host, which can be ananimal, such as a mammal. Examples of cells include, but are not limitedto, a human cell, a human cell line, E. coli (e.g., E. coli TB-1, TG-2,DH5a, XL-Blue MRF′ (Stratagene), SA2821 and Y1090), B. subtilis, P.aerugenosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9. Ea4) andothers set forth herein below. In a preferred embodiment, the cell is aCHO cell. In an especially preferred embodiment, the cell is a CHO-Scell.

In certain embodiments, a polynucleotide described herein can beexpressed in a stable cell line that comprises the polynucleotideintegrated into a chromosome by introducing the polynucleotide into thecell. In certain embodiments, the polynucleotide is introduced into thecell by, for example, electroporation. In certain embodiments, thepolynucleotide is introduced into the cell by, for example, transfectionof a vector comprising the polynucleotide into the cell. In certainembodiments, the vector is co-transfected with a selectable marker suchas DHFR, GPT, neomycin, or hygromycin to allow for the identificationand isolation of the transfected cells. In certain embodiments, thetransfected polynucleotide can also be amplified to express largeamounts of the encoded bispecific binding molecule. For example, theDHFR (dihydrofolate reductase) marker can be utilized to develop celllines that carry several hundred or even several thousand copies of thepolynucleotide of interest. Another example of a selection marker is theenzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279(1991); Bebbington, et al., Bio/Technology 10:169-175 (1992)). Usingthese markers, the cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of bispecificbinding molecules.

In a preferred embodiment, the vector comprises (i) a firstpolynucleotide sequence encoding a light chain fusion polypeptidecomprising an immunoglobulin light chain fused to a scFv, via a peptidelinker, wherein the light chain binds to HER2 and wherein the scFv bindsto CD3, operably linked to a first promoter and (ii) a secondpolynucleotide encoding an immunoglobulin heavy chain that binds to HER2operably linked to a second promoter. In certain embodiments, the vectoris a viral vector.

5.4 T Cells Bound to Bispecific Binding Molecules

Without being bound by any theory, it is believed that when thebispecific binding molecules provided herein are bound to T cells, by,for example, procedures such as those described herein, an anti-CD3 scFvof the bispecific binding molecule binds to CD3 on the surface of the Tcell. Without being bound by any theory, it is believed that binding ofthe bispecific binding molecule to the T cell (i.e., binding of ananti-CD3 scFv to CD3 expressed on the T cell) activates the T cell, andconsequently, allows for the T cell receptor-based cytotoxicity to beredirected to desired tumor targets, bypassing MHC restrictions.

Thus, the invention also provides T cells which are bound to abispecific binding molecule of the invention (e.g., as described inSection 5.1 and Section 5.2). In specific embodiments, the T cells arebound to the bispecific binding molecule noncovalently. In specificembodiments, the T cells are autologous to a subject to whom the T cellsare to be administered. In specific embodiments, the T cells areallogeneic to a subject to whom the T cells are to be administered. Inspecific embodiments, the T cells are human T cells.

In specific embodiments, the T cells which are bound to bispecificbinding molecules of the invention are used in accordance with themethods described in Section 5.6. In specific embodiments, the T cellswhich are bound to bispecific binding molecules of the invention areused as part of a combination therapy as described in Section 5.9.

5.5 Pharmaceutical Compositions and Kits

In certain embodiments, provided herein are compositions (e.g.,pharmaceutical compositions) and kits comprising a pharmaceuticallyeffective amount of one or more bispecific binding molecule as describedin Section 5.1 or Section 5.2. Compositions may be used in thepreparation of individual, single unit dosage forms. Compositionsprovided herein can be formulated for parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intra-Ommaya,intraocular, intravitreous, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, intrathecal,intraventricular in the brain, intraparenchymal in the brain, ortransdermal administration. In a preferred embodiment, the compositionis formulated for parenteral administration. In an especially preferredembodiment, the composition is formulated for intravenousadministration. In a preferred embodiment, the composition is formulatedfor intraperitoneal administration. In a specific embodiment, thecomposition is formulated for intraperitoneal administration to treatperitoneal metastases. In a preferred embodiment, the composition isformulated for intrathecal administration. In a specific embodiment, thecomposition is formulated for intrathecal administration to treat brainmetastases. See, for example, Kramer et al., 2010, 97: 409-418. In apreferred embodiment, the composition is formulated for intraventricularadministration in the brain. In a specific embodiment, the compositionis formulated for intraventricular administration to treat brainmetastases. See, for example, Kramer et al., 2010, 97: 409-418. In apreferred embodiment, the composition is formulated for intraparenchymaladministration in the brain. In a specific embodiment, the compositionis formulated for intraparenchymal administration to treat a brain tumoror brain tumor metastases. See, for example, Luther et al., 2014, NeuroOncol, 16: 800-806, and Clinical Trial ID NO NCT01502917.

In a specific embodiment, the composition is formulated forintraperitoneal administration for peritoneal metastases.

In certain embodiments, provided herein is a composition comprising oneor more polynucleotide comprising nucleotide sequences encoding abispecific binding molecule as described herein. In certain embodiments,provided herein is a composition comprising a cell, wherein the cellcomprises one or more polynucleotide comprising nucleotide sequencesencoding a bispecific binding molecule as described herein. In certainembodiments, provided herein is a composition comprising a vector,wherein the vector comprises one or more polynucleotide comprisingnucleotide sequences encoding a bispecific binding molecule as describedherein. In certain embodiments, provided herein is a compositioncomprising a cell, wherein the cell comprises a vector, wherein thevector comprises one or more polynucleotide comprising nucleotidesequences encoding a bispecific binding molecule as described herein.

In certain embodiments, a composition described herein is a stable orpreserved formulation. In certain embodiments, the stable formulationcomprises a phosphate buffer with saline or a chosen salt. In certainembodiments, a composition described is a multi-use preservedformulation, suitable for pharmaceutical or veterinary use. In certainembodiments, a composition described herein comprises a preservative.Preservatives are known to one of ordinary skill in the art.Non-limiting examples of preservatives include phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuricnitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride(e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and thelike), benzalkonium chloride, benzethonium chloride, and sodiumdehydroacetate and thimerosal, or mixtures thereof in an aqueousdiluent. Any suitable concentration or mixture can be used as known inthe art, such as 0.001-5%, or any range or value therein, such as, butnot limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5,4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examplesinclude, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5,0.9, 1.0/), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0,2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol(e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s)(e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02,0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

It can be sometimes desirable to deliver the compositions providedherein to a subject over prolonged periods of time, for example, forperiods of one week to one year or more from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g., a zinc tannate salt. Additionally, a compositionprovided herein, preferably, a relatively insoluble salt such as thosejust described, can be formulated in a gel, for example, an aluminummonostearate gel with, e.g., sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer, for example, as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant compostions, e.g., gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

The range of at least one bispecific binding molecule compositionprovided herein includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 microgram/ml to about 1000mg/ml, although lower and higher concentrations are operable and aredependent on the intended delivery vehicle, e.g., solution formulationswill differ from transdermal patch, pulmonary, transmucosal, or osmoticor micro pump methods.

In certain embodiments, compositions provided herein comprise at leastone of any suitable auxiliary, such as, but not limited to, diluent,binder, stabilizer, buffers, salts, lipophilic solvents, preservative,adjuvant or the like. In certain embodiments, pharmaceuticallyacceptable auxiliaries are preferred. Non-limiting examples of, andmethods of preparing such sterile solutions are well known in the art,such as, but not limited to, Gennaro, Ed., Remington's PharmaceuticalSciences, 18^(th) Edition, Mack Publishing Co. (Easton, Pa.) 1990.Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofthe bispecific binding molecule as described herein.

In certain embodiments, compositions provided herein contain one or morepharmaceutical excipient and/or additive. Non-limiting examples ofpharmaceutical excipients and additives are proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Non-limiting examples of protein excipients include serumalbumin such as human serum albumin (HSA), recombinant human albumin(rHA), gelatin, casein, and the like. Non-limiting examples of aminoacid/antibody components, which can also function in a bufferingcapacity, include alanine, glycine, arginine, betaine, histidine,glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine,valine, methionine, phenylalanine, aspartame, and the like. In certainembodiments, the amino acid is glycine. Non-limiting examples ofcarbohydrate excipients include monosaccharides such as fructose,maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositoland the like. In certain embodiments, the carbohydrate excipient ismannitol, trehalose, or raffinose.

In certain embodiments, a composition provided herein includes one ormore buffer or a pH adjusting agent; typically, the buffer is a saltprepared from an organic acid or base. Non-limiting examples of buffersinclude organic acid salts such as salts of citric acid, ascorbic acid,gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid; Tris, tromethamine hydrochloride, or phosphatebuffers. In certain embodiments, the buffer is an organic acid saltssuch as citrate. Other excipients, e.g., isotonicity agents, buffers,antioxidants, preservative enhancers, can be optionally and preferablyadded to the diluent. An isotonicity agent, such as glycerin, iscommonly used at known concentrations. A physiologically toleratedbuffer is preferably added to provide improved pH control. Thecompositions can cover a wide range of pHs, such as from about pH 4 toabout pH 10, and preferred ranges from about pH 5 to about pH 9, and amost preferred range of about 6.0 to about 8.0. Preferably, thecompositions provided herein have pH between about 6.8 and about 7.8.Preferred buffers include phosphate buffers, most preferably sodiumphosphate, particularly phosphate buffered saline (PBS).

In certain embodiments, a composition provided herein includes one ormore polymeric excipient/additive such as, for example,polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin),polyethylene glycols, flavoring agents, antimicrobial agents,sweeteners, antioxidants, antistatic agents, surfactants (e.g.,polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g.,phospholipids, fatty acids), steroids (e.g., cholesterol), and/orchelating agents (e.g., EDTA).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornonionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the compositions to reduceaggregation. These additives are particularly useful if a pump orplastic container is used to administer the composition. The presence ofpharmaceutically acceptable surfactant mitigates the propensity for theprotein to aggregate.

Additional pharmaceutical excipients and/or additives suitable for usein a composition provided herein are known to one of skill in the artand are referenced in, for example, “Remington: The Science & Practiceof Pharmacy”, 19.sup.th ed., Williams & Williams, (1995), and in the“Physician's Desk Reference”, 52^(nd) ed., Medical Economics, Montvale,N.J. (1998), which are entirely incorporated herein by reference. Incertain preferred embodiments, the carrier or excipient materials arecarbohydrates (e.g., saccharides and alditols) and buffers (e.g.,citrate) or polymeric agents.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the composition is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

The compositions provided herein can be prepared by a process whichcomprises mixing at least one bispecific binding molecule as describedherein and a preservative selected from the group consisting of phenol,m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal ormixtures thereof in an aqueous diluent. Mixing the at least onebispecific binding molecule and preservative in an aqueous diluent iscarried out using conventional dissolution and mixing procedures. Toprepare a suitable composition, for example, a measured amount of atleast one bispecific binding molecule in buffered solution is combinedwith the desired preservative in a buffered solution in quantitiessufficient to provide the bispecific binding molecule and preservativeat the desired concentrations. The compositions provided herein can beprepared by a process that comprises mixing at least one bispecificbinding molecule as described herein and a selected buffer, preferably aphosphate buffer containing saline or a chosen salt. Mixing the at leastone bispecific binding molecule and buffer in an aqueous diluent iscarried out using conventional dissolution and mixing procedures. Toprepare a suitable composition, for example, a measured amount of atleast one bispecific binding molecule in water or buffer is combinedwith the desired buffering agent in water in quantities sufficient toprovide the protein and buffer at the desired concentrations. Variationsof these processes would be recognized by one of ordinary skill in theart. For example, the order the components are added, whether additionaladditives are used, the temperature and pH at which the composition isprepared, are all factors that can be optimized for the concentrationand means of administration used.

In specific embodiments involving combination therapy with infusion of Tcells, provided herein is a pharmaceutical composition comprising (a) abispecific binding molecule described herein (see, e.g., Section 5.1 or5.2); (b) T cells; and/or (c) a pharmaceutically effective carrier. Inspecific embodiments, the T cells are autologous to the subject to whomthe T cells are administered. In certain embodiments, the T cells areallogeneic to the subject to whom the T cells are administered. Inspecific embodiments, the T cells are bound to the bispecific bindingmolecule. In specific embodiments, the binding of the T cells to thebispecific binding molecule is noncovalently. In specific embodiments,the T cells are human T cells. Methods that can be used to bindbispecific binding molecules to T cells are known in the art. See, e.g.,Lum et al., 2013, Biol Blood Marrow Transplant, 19:925-33, Janeway etal., Immunobiology: The Immune System in Health and Disease, 5^(1h)edition, New York: Garland Science; Vaishampayan et al., 2015, ProstateCancer, 2015:285193, and Stromnes et al., 2014, Immunol Rev.257(1):145-164. See, also, Vaishampayan et al., 2015, Prostate Cancer,2015:285193, which describes the following exemplary, non-limitingmethod for binding bispecific binding molecules to T cells:

-   -   Peripheral blood mononuclear cells (PBMCs) are collected to        obtain lymphocytes for activated T cell expansion from 1 or 2        leukopheresis. PBMCs are activated with, for example, 20 ng/mL        of OKT3 and expanded in 100 IU/mL of IL-2 to generate 40-320        billion activated T cells during a maximum of 14 days of culture        under cGMP conditions as described in Ueda et al., 1993,        Transplantation, 56(2):351-356 and Uberti et al., 1994, Clinical        Immunology and Immunopathology, 70(3):234-240. Cells are grown        in breathable flasks (FEP Bag Type 750-C1, American Fluoroseal        Corporation, Gaithersburg, Md.) in RPMI 1640 medium (Lonza)        supplemented with 2% pooled heat inactivated human serum.        Activated T cells are split approximately every 2-3 days based        on cell counts. After 14 days, activated T cells are cultured        with 50 ng of a bispecific binding molecule described herein per        10⁶ activated T cells. The mixture is then washed and        cryopreserved.

In certain embodiments, a pharmaceutical composition described herein isto be used in accordance with the methods provided herein (see, e.g.,Section 5.6).

5.5.1 Parenteral Formulations

In certain embodiments, a composition provided herein is formulated forparenteral injectable administration. As used herein, the term“parenteral” includes intravenous, intravascular, intramuscular,intradermal, subcutaneous, and intraocular. For parenteraladministration, the composition can be formulated as a solution,suspension, emulsion or lyophilized powder in association, or separatelyprovided, with a pharmaceutically acceptable parenteral vehicle.Non-limiting examples of such vehicles are water, saline, Ringer'ssolution, dextrose solution, glycerol, ethanol, and 1-10% human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils can alsobe used. The vehicle or lyophilized powder can contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthetic mono- or di- or tri-glycerides. Parental administration isknown in the art and includes, but is not limited to, conventional meansof injections, a gas pressured needle-less injection device as describedin U.S. Pat. No. 5,851,198, and a laser perforator device as describedin U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

5.5.2 Pulmonary Formulations

In certain embodiments, a composition comprising a bispecific bindingmolecule described herein is formulated for pulmonary administration.For pulmonary administration, the composition is delivered in a particlesize effective for reaching the lower airways of the lung or sinuses.Compositions for pulmonary administration can be delivered by any of avariety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of bispecific bindingmolecules described herein are also known in the art. All such devicesuse formulations suitable for the administration for the dispensing of abispecific binding molecule described herein in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler®. (Glaxo), Diskus®(Glaxo), devices marketed by Inhale Therapeutics, to name a few, usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like the Ultravent® nebulizer (Mallinckrodt), andthe Acorn II® nebulizer (Marquest Medical Products) (U.S. Pat. No.5,404,871 Aradigm, WO 97/22376), the above references entirelyincorporated herein by reference, produce aerosols from solutions, whilemetered dose inhalers, dry powder inhalers, etc. generate small particleaerosols. Such examples of commercially available inhalation devices arenon-limiting examples are not intended to be limiting in scope.

In certain embodiments, a spray comprising a bispecific binding moleculeas described herein can be produced by forcing a suspension or solutionof at least one bispecific binding molecule as described herein througha nozzle under pressure. The nozzle size and configuration, the appliedpressure, and the liquid feed rate can be chosen to achieve the desiredoutput and particle size. An electrospray can be produced, for example,by an electric field in connection with a capillary or nozzle feed.Advantageously, particles of a composition comprising at least onebispecific binding molecule described herein delivered by a sprayer havea particle size less than about 10 um, preferably in the range of about1 um to about 5 um, and most preferably about 2 um to about 3 um.

Formulations of a composition comprising at least one bispecific bindingmolecule described herein suitable for use with a sprayer typicallyinclude the at least one bispecific binding molecule in an aqueoussolution at a concentration of about 0.1 mg to about 100 mg per ml ofsolution or mg/gm, or any range or value therein, e.g., but not limitedto, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. Theformulation can include agents such as an excipient, a buffer, anisotonicity agent, a preservative, a surfactant, and, preferably, zinc.The formulation can also include an excipient or agent for stabilizationof the bispecific binding molecule composition, such as a buffer, areducing agent, a bulk protein, or a carbohydrate. Bulk proteins usefulin formulating such a composition include albumin, protamine, or thelike. Typical carbohydrates useful in formulating antibody compositionproteins include sucrose, mannitol, lactose, trehalose, glucose, or thelike. The composition can also include a surfactant, which can reduce orprevent surface-induced aggregation of the composition caused byatomization of the solution in forming an aerosol. Various conventionalsurfactants can be employed, such as polyoxyethylene fatty acid estersand alcohols, and polyoxy ethylene sorbitol fatty acid esters. Amountswill generally range between 0.001 and 14% by weight of the formulation.Preferred surfactants are polyoxyethylene sorbitan monooleate,polysorbate 80, polysorbate 20, or the like.

In certain embodiments, the composition is administered via a nebulizer,such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jetnebulizer, a compressed air source is used to create a high-velocity airjet through an orifice. As the gas expands beyond the nozzle, alow-pressure region is created, which draws a solution of antibodycomposition protein through a capillary tube connected to a liquidreservoir. The liquid stream from the capillary tube is sheared intounstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of antibody composition protein either directly or through acoupling fluid, creating an aerosol including the antibody compositionprotein. Advantageously, particles of antibody composition proteindelivered by a nebulizer have a particle size less than about 10 um,preferably in the range of about 1 um to about 5 um, and most preferablyabout 2 um to about 3 um.

In certain embodiments, the composition is administered via a metereddose inhaler (MDI), wherein a propellant, at least one bispecificbinding molecule described herein, and any excipients or other additivesare contained in a canister as a mixture including a liquefiedcompressed gas. Actuation of the metering valve releases die mixture asan aerosol, preferably containing particles in the size range of lessthan about 10 um, preferably about 1 um to about 5 um, and mostpreferably about 2 um to about 3 um. The desired aerosol particle sizecan be obtained by employing a formulation of antibody compositionprotein produced by various methods known to those of skill in the art,including jet-milling, spray drying, critical point condensation, or thelike. Preferred metered dose inhalers include those manufactured by 3Mor Glaxo and employing a hydrofluorocarbon propellant.

Formulations of a bispecific binding molecule described herein for usewith a metered-dose inhaler device will generally include a finelydivided powder containing at least one Anti-IL-6 antibody as asuspension in a non-aqueous medium, for example, suspended in apropellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one bispecific binding molecule as asuspension in the propellant, to protect the active agent againstchemical degradation, and the like. Suitable surfactants includesorbitan trioleate, soya lecithin, oleic acid, or the like. In somecases solution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation.

5.5.3 Oral Formulations

In certain embodiments, a composition provided herein is formulated fororal administration. In certain embodiments, for oral administration,compositions and methods of administering at least one bispecificbinding molecule described herein rely on the co-administration ofadjuvants such as, for example, resorcinols and nonionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether,to artificially increase the permeability of the intestinal walls, aswell as the co-administration of enzymatic inhibitors such as, forexample, pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF)and trasylol, to inhibit enzymatic degradation. The active constituentcompound of the solid-type dosage form for oral administration can bemixed with at least one additive, including sucrose, lactose, cellulose,mannitol, trehalose, raffinose, maltitol, dextran, starches, agar,arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic,gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer,and glyceride. These dosage forms can also contain other type(s) ofadditives, such as, for example, inactive diluting agent, lubricant suchas magnesium stearate, paraben, preserving agent such as sorbic acid,ascorbic acid, alpha.-tocopherol, antioxidant such as cysteine,disintegrator, binder, thickener, buffering agent, sweetening agent,flavoring agent, perfuming agent, etc.

In certain embodiments, tablets and pills for oral administration can befurther processed into enteric-coated preparations. In certainembodiments, liquid preparations for oral administration include, forexample, emulsion, syrup, elixir, suspension and solution preparationsallowable for medical use. These preparations can contain inactivediluting agents ordinarily used in said field, for example, water.Liposome preparations can be utilized for oral administrationpreparations, for example, as described for insulin and heparin (U.S.Pat. No. 4,239,754). Additionally, microspheres of artificial polymersof mixed amino acids (proteinoids) can be utilized to in oraladministration of pharmaceuticals, for example, as described in U.S.Pat. No. 4,925,673. Furthermore, carrier compounds, such as thosedescribed in U.S. Pat. Nos. 5,879,681 and 5,871,753, are used in oraladministration of biologically active agents.

5.5.4 Mucosal Formulations

In certain embodiments, a composition provided herein is formulated forabsorption through mucosal surfaces. In certain embodiments, forabsorption through mucosal surfaces, compositions and methods ofadministering at least one bispecific binding molecule described hereininclude an emulsion comprising a plurality of submicron particles, amucoadhesive macromolecule, a bioactive peptide, and an aqueouscontinuous phase, which promotes absorption through mucosal surfaces byachieving mucoadhesion of the emulsion particles (U.S. Pat. No.5,514,670). Mucous surfaces suitable for application of the emulsionsprovided herein can include, for example, corneal, conjunctival, buccal,sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectalroutes of administration. Formulations for vaginal or rectaladministration, for example, suppositories, can contain as excipients,for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.Formulations for intranasal administration can be solid and contain asexcipients, for example, lactose or can be aqueous or oily solutions ofnasal drops. For buccal administration excipients include, for example,sugars, calcium stearate, magnesium stearate, pregelinatined starch, andthe like (U.S. Pat. No. 5,849,695).

5.5.5 Transdermal Formulations

In certain embodiments, a composition provided herein is formulated fortransdermal administration. In certain embodiments, for transdermaladministration, the composition comprises at least one bispecificbinding molecule described herein encapsulated in a delivery device suchas, for example, a liposome or polymeric nanoparticles, microparticle,microcapsule, or microspheres (referred to collectively asmicroparticles unless otherwise stated). A number of suitable devicesare known for transdermal administration, including microparticles madeof synthetic polymers such as polyhydroxy acids such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

5.5.6 Kits

Provided herein are kits comprising one or more bispecific bindingmolecule as described herein, or one or more composition as describedherein. In certain embodiments, the kit comprises packaging material andat least one vial comprising a composition comprising a bispecificbinding molecule or composition described herein. In certainembodiments, the vial comprises a solution of at least one bispecificbinding molecule or composition as described herein with the prescribedbuffers and/or preservatives, optionally in an aqueous diluents. Incertain embodiments, the packaging material comprises a label thatindicates that such solution can be held over a period of 1, 2, 3, 4, 5,6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.In certain embodiments, the kit comprises two vials. In certainembodiments, the first vial comprises at least one lyophilizedbispecific binding molecule or composition as described herein and thesecond vial comprises aqueous diluents of prescribed buffer orpreservative. In certain embodiments, the packaging material comprises alabel that instructs a subject to reconstitute the at least onelyophilized bispecific binding molecule in the aqueous diluents to forma solution that can be held over a period of twenty-four hours orgreater. In certain embodiments, the packaging material comprises alabel that indicates that such solution can be held over a period of 1,2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hoursor greater.

In certain embodiments, the compositions provided herein can be providedto a subject as solutions or as dual vials comprising a vial oflyophilized at least one bispecific binding molecule or composition thatis reconstituted with a second vial containing water, a preservativeand/or excipients, preferably a phosphate buffer and/or saline and achosen salt, in an aqueous diluent. Either a single solution vial ordual vial requiring reconstitution can be reused multiple times and cansuffice for a single or multiple cycles of subject treatment and thuscan provide a more convenient treatment regimen than currentlyavailable.

In certain embodiments, a kit comprising a bispecific binding moleculeor composition described herein is useful for administration over aperiod of immediately to twenty-four hours or greater. Accordingly, thekit offers significant advantages to the patient. In certainembodiments, a kit comprising a bispecific binding molecule orcomposition described herein can optionally be safely stored attemperatures of from about 2° C. to about 40° C. and retain thebiologically activity of the protein for extended periods of time, thus,allowing a package label indicating that the solution can be held and/orused over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.In certain embodiments, the kit comprises a

If preserved diluent is used, such label can include use up to 1-12months, one-half, one and a half, and/or two years.

The kits can be provided indirectly to a subject, such as a subject asdescribed in Section 5.7, by providing to pharmacies, clinics, or othersuch institutions and facilities, solutions or dual vials comprising avial of lyophilized at least one bispecific binding molecule orcomposition that is reconstituted with a second vial containing theaqueous diluent. The solution in this case can be up to one liter oreven larger in size, providing a large reservoir from which smallerportions of the at least one antibody solution can be retrieved one ormultiple times for transfer into smaller vials and provided by thepharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject®, e.g., as made or developed by Becton Dickensen(Franklin Lakes, N.J.,), Disetronic (Burgdorf, Switzerland; Bioject,Portland, Oreg.; National Medical Products, Weston Medical(Peterborough, UK), Medi-Ject Corp (Minneapolis, Minn.). Recognizeddevices comprising a dual vial system include those pen-injector systemsfor reconstituting a lyophilized drug in a cartridge for delivery of thereconstituted solution such as the HumatroPen®.

In certain embodiments, the kits comprise packaging material. In certainembodiments, the packaging material provides, in addition to theinformation required by a regulatory agencies, the conditions underwhich the product can be used. In certain embodiments, the packagingmaterial provides instructions to the subject to reconstitute the atleast one bispecific binding molecule in the aqueous diluent to form asolution and to use the solution over a period of 2-24 hours or greaterfor the two vial, wet/dry, product. For the single vial, solutionproduct, the label indicates that such solution can be used over aperiod of 2-24 hours or greater. In a preferred embodiment, the kit isuseful for human pharmaceutical product use. In certain embodiments, thekit is useful for veterinarian pharmaceutical use. In a preferredembodiment, the kit is useful for canine pharmaceutical product use. Ina preferred embodiment, the kit is useful for intravenousadministration. In another preferred embodiment, the kit is useful forintraperitoneal, intrathecal, intraventricular in the brain, orintraparenchymal in the brain administration.

5.6 Uses and Methods 5.6.1 Therapeutic Uses

In certain embodiments, provided herein are methods for treating aHER2-positive cancer in a subject comprising administering to thesubject in need thereof a therapeutically effective amount of abispecific binding molecule as described in Section 5.1 or in Section5.2, a therapeutically effective amount of a cell, polynucleotide, orvector encoding such a bispecific binding molecule as described inSection 5.3, or a therapeutically effective amount of a pharmaceuticalcomposition as described in Section 5.5, or a therapeutically effectiveamount of T cells bound to a bispecific binding molecule as described inSection 5.4. In a specific embodiment, the subject is a subject asdescribed in Section 5.7. In a specific embodiment, the bispecificbinding molecule is administered at a dose as described in Section 5.8.In a specific embodiment, the bispecific binding molecule isadministered according to the methods as described in Section 5.5. In apreferred embodiment, the bispecific binding molecule is administeredintravenously. In another preferred embodiment, the bispecific bindingmolecule is administered intrathecally, intraventricularly in the brain,intraparenchymally in the brain, or intraperitoneally. In a specificembodiment, the bispecific binding molecule is administered incombination with one or more additional pharmaceutically active agentsas described in Section 5.9.

In certain embodiments, provided herein are methods for treating aHER2-positive cancer in a subject comprising administering to thesubject in need thereof a pharmaceutical composition as described inSection 5.1 or in Section 5.2. In a specific embodiment, thepharmaceutical composition is a composition as described in Section 5.5.In a specific embodiment, the subject is a subject as described inSection 5.7. In a specific embodiment, the pharmaceutical composition isadministered at a dose as described in Section 5.8. In a specificembodiment, the pharmaceutical composition is administered according tothe methods as described in Section 5.5. In a preferred embodiment, thepharmaceutical composition is administered intravenously. In anotherpreferred embodiment, the bispecific binding molecule is administeredintrathecally, intraventricularly in the brain, intraparenchymally inthe brain, or intraperitoneally. In a specific embodiment, thepharmaceutical composition is administered in combination with one ormore additional pharmaceutically active agents as described in Section5.9.

For use of a bispecific binding molecule in a subject of a particularspecies, a bispecific binding molecule is used that binds to the HER2and the CD3 of that particular species. For example, to treat a human,the bispecific binding molecule comprises an aglycosylated monoclonalantibody that is an immunoglobulin that binds to human HER2, comprisingtwo identical heavy chains and two identical light chains, said lightchains being a first light chain and a second light chain, wherein thefirst light chain is fused to a first single chain variable fragment(scFv), via a peptide linker, to create a first light chain fusionpolypeptide, and wherein the second light chain is fused to a secondscFv, via a peptide linker, to create a second light chain fusionpolypeptide, wherein the first and second scFv (i) are identical, and(ii) bind to human CD3, and wherein the first and second light chainfusion polypeptides are identical. In another example, to treat acanine, the bispecific binding molecule comprises an aglycosylatedmonoclonal antibody that is an immunoglobulin that binds to canine HER2,comprising two identical heavy chains and two identical light chains,said light chains being a first light chain and a second light chain,wherein the first light chain is fused to a first single chain variablefragment (scFv), via a peptide linker, to create a first light chainfusion polypeptide, and wherein the second light chain is fused to asecond scFv, via a peptide linker, to create a second light chain fusionpolypeptide, wherein the first and second scFv (i) are identical, and(ii) bind to canine CD3, and wherein the first and second light chainfusion polypeptides are identical. Bispecific binding molecules that arecross-reactive with HER2 and/or CD3 of various species can be used totreat subjects in those species. For example, trastuzumab is expected tobind both human and canine HER2 due to the relative conservation of theepitope in HER2 recognized by trastuzumab. See, also, for example,Singer et al., 2012, Mol Immunol, 50: 200-209.

In addition, for use of a bispecific binding molecule in a subject of aparticular species, the bispecific binding molecule, preferably, theconstant region of the immunoglobulin portion, is derived from thatparticular species. For example, to treat a human, the bispecificbinding molecule can comprise an aglycosylated monoclonal antibody thatis an immunoglobulin, wherein the immunoglobulin comprises a humanconstant region. In another example, to treat a canine, the bispecificbinding molecule can comprise an aglycosylated monoclonal antibody thatis an immunoglobulin, wherein the immunoglobulin comprises a canineconstant region. In a specific embodiment, when treating a human, theimmunoglobulin is humanized. In a specific embodiment, the subject is ahuman. In a specific embodiment, the subject is a canine.

In a specific embodiment, the HER2-positive cancer is breast cancer,gastric cancer, an osteosarcoma, desmoplastic small round cell cancer,squamous cell carcinoma of head and neck cancer, ovarian cancer,prostate cancer, pancreatic cancer, glioblastoma multiforme, gastricjunction adenocarcinoma, gastroesophageal junction adenocarcinoma,cervical cancer, salivary gland cancer, soft tissue sarcoma, leukemia,melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any otherneoplastic tissue that expresses the HER2 receptor.

In a specific embodiment, the HER2-positive cancer is resistant totreatment with trastuzumab, cetuximab, lapatinib, erlotinib, or anyother small molecule or antibody that targets the HER family ofreceptors. In a specific embodiment, the tumor that is resistant totreatment with trastuzumab, cetuximab, lapatinib, erlotinib, or anyother small molecule or antibody that targets the HER family ofreceptors is responsive to treatment with a bispecific binding moleculeof the invention. In a specific embodiment, the HER2-positive cancer isresistant to treatment with necitumumab, pantitumumab, pertuzumab, orado-trastuzumab emtansine. In a specific embodiment, the HER2-positivecancer that is resistant to treatment with necitumumab, pantitumumab,pertuzumab, or ado-trastuzumab emtansine is responsive to treatment witha bispecific binding molecule of the invention.

In a specific embodiment, the HER2-positive cancer is a cancer thatexpresses programmed death-ligand 1 (“PDL1” or “PDL-1”) (i.e., aHER2-positive, PDL1-positive cancer). Thus, in a specific embodiment,provided herein is a method of treating a HER2-positive, PDL1-positivecancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a bispecific bindingmolecule as described in Section 5.1 or in Section 5.2, atherapeutically effective amount of a cell, polynucleotide, or vectorencoding such a bispecific binding molecule as described in Section 5.3,or a therapeutically effective amount of a pharmaceutical composition asdescribed in Section 5.5, or a therapeutically effective amount of Tcells bound to a bispecific binding molecule as described in Section5.4. In a specific embodiment, the HER2-positive, PDL1-positive canceroverexpresses PDL1. In a specific embodiment, the HER2-positive,PDL1-positive cancer overexpresses PDL1 in cancerous cells relative toexpression of PDL1 in analogous noncancerous cells of the same tissuetype as the HER2-positive, PDL1-positive cancer. The noncancerous cellsare analogous to the cancerous cells by virtue of the fact that they,for example, are from the same tissue or organ type or are otherwisesuitable for comparison of PDL1 expression. The level of PDL1 expressionin analogous noncancerous cells can be a known, standard level for apopulation or for particular individual(s) or for the subject havingcancer, or can be newly measured. The overexpression can be shown, forexample, by detecting increased PDL1 expression in a test specimencomprising cancerous cells relative to expression in a control specimencomprising analogous noncancerous cells. In contrast to the testspecimen, the control specimen does not contain a significant amount ofcancerous cells. In a specific embodiment, a HER2-positive,PDL1-positive cancer is deemed to overexpress PDL1 when the testspecimen expresses a detectable level of PDL1 above background (i.e.,experimental noise), preferably as measured by immunohistochemistry(“IHC”) since most normal tissue should be PDL1-negative. In a specificembodiment, the detectable level of PDL1 above background is 1% to 5%,or is at least 1%, at least 2%, at least 3%, at least 4%, or at least 5%above background. In a specific embodiment in which the HER2-positive,PDL1-positive cancer is a melanoma, in a particular embodiment, themelanoma is deemed to overexpress PDL1 when the test specimen expressesa detectable level of PDL1 that is at least 5% above background. In aspecific embodiment in which the HER2-positive, PDL1-positive cancer isa non-small cell lung carcinoma, in a particular embodiment, thenon-small cell lung carcinoma is deemed to overexpress PDL1 if the testspecimen expresses a detectable level of PDL1 that is at least 5% abovebackground. In a specific embodiment where binding to an anti-PDL1antibody is used to measure the level of PDL1 expression in the testspecimen and the control specimen, in a particular embodiment, thebackground level is measured by measuring nonspecific signal, forexample, arising from binding to an antibody that recognizes an antigenknown not to be expressed by the test or control specimen, e.g., ananti-IgG antibody. In a specific embodiment, PDL1 expression is measuredby measuring PDL1 protein levels. In a specific embodiment, PDL1expression is measured by measuring PDL1 nucleic acid levels (e.g., cDNAor RNA encoding PDL1). In a specific embodiment, PDL1 protein level ismeasured according to any assay known in the art, such as, e.g., IHC,western blot, enzyme-linked immunosorbent assay, orfluorescence-activated cell sorting. In a specific embodiment, PDL1nucleic acid level is measured according to any assay known in the art,such as, e.g., in situ hybridization (“ISH”), southern blot, northernblot, quantitative reverse transcriptase polymerase chain reaction, ordeep sequencing. The test specimen comprises cancer cells from thesubject having cancer, and may be in the form of various biologicalspecimens known in the art, e.g., from a biopsy or surgical resection.In a specific embodiment, a test specimen comprises cancerous cells froma primary tumor from the subject having cancer. In a specificembodiment, the test specimen comprises cancerous cells from ametastatic tumor from the subject having cancer. In a specificembodiment, the control specimen comprising analogous noncancerous cells(analogous to the cancerous cells in the test specimen) is a specimenobtained or derived from the subject who has cancer. Alternatively, acontrol specimen may be a specimen obtained or derived from a healthysubject or a subject who does not have cancer. In a specific embodiment,the control specimen does not comprise cancerous cells. In a specificembodiment, the test specimen and control specimen are from the samesubject. In a specific embodiment, the test specimen and the controlspecimen are from different subjects. In a specific embodiment, the testspecimen contains cancerous cells from breast tissue and the controlspecimen contains noncancerous cells from breast tissue.

Nonlimiting examples of HER2-positive cancers that express PDL1 and thuscan be treated according to the methods described herein include breastcancer, gastric cancer, an osteosarcoma, desmoplastic small round cellcancer, ovarian cancer, prostate cancer, pancreatic cancer, glioblastomamultiforme, gastric junction adenocarcinoma, gastroesophageal junctionadenocarcinoma, cervical cancer, salivary gland cancer, soft tissuesarcoma, leukemia, melanoma, Ewing's sarcoma, rhabdomyosarcoma, orneuroblastoma. In a specific embodiment, the HER2-positive,PDL1-positive cancer is not a head and neck cancer.

In a specific embodiment, a HER2-positive, PDL1-positive cancer treatedaccording to the methods described herein is resistant to PDL1 blockadewith an anti-PDL1 therapy. In a specific embodiment, the HER2-positive,PDL1-positive cancer is resistant to programmed cell death 1 (“PD1” or“PD-1”) blockade with an anti-PD1 therapy. In a specific embodiment, theHER2-positive, PDL1-positive cancer is resistant to (i) PDL1 blockadewith an anti-PDL1 therapy, and (ii) PD1 blockade with an anti-PD1therapy.

In a specific embodiment, PDL1 blockade refers to (i) inhibition ofPDL1-dependent PD1 activation, or (ii) blocking of PDL1 binding to PD1.In a specific embodiment, the inhibition or blocking is partial. Inanother specific embodiment, the inhibition or blocking is complete. Ina specific embodiment, PDL1 blockade refers to least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% inhibition of PDL1-dependent PD1 activation as assessedby any method known to one of skill in the art, such as, e.g., aphosphorylation assay, as compared to PDL1-dependent PD1 activation inthe presence of a negative control therapy (e.g., an anti-IgG antibody).For example, in a specific embodiment, PDL1 blockade that is inhibitionof activation is assessed by (a) contacting a PDL1-expressing cell and aPD1-expressing activated T cell with an anti-PDL1 therapy (e.g., ananti-PDL1 antibody) or a negative control therapy (e.g., an anti-IgGantibody), and (b) measuring the phosphorylation of PD1 ordephosphorylation of a downstream signaling molecule, such as, e.g., Lckor Zap-70, as assessed by, for e.g., ELISA or western blot, in thepresence of the anti-PDL1 therapy as compared to the phosphorylation ofPD1 or dephosphorylation of a downstream signaling molecule, such as,e.g., Lck or Zap-70, as assessed by, for e.g., ELISA or western blot, inthe presence of the negative control therapy. In a specific embodiment,PDL1 blockade that is blocking of PDL1 binding to PD1 is assessed by (a)contacting a PDL1-expressing cell and a PD1-expressing activated T cellwith an anti-PDL1 therapy (e.g., an anti-PDL1 antibody) or a negativecontrol therapy (e.g., an anti-IgG antibody), and (b) measuring theinteraction between PDL1 and PD1 by, for example, co-localization (asassessed by, e.g., immunohistochemistry) or co-immunoprecipitation (asassessed by, e.g., western blot) of PDL1 and PD1, in the presence of theanti-PDL1 therapy as compared to the interaction between PDL1 and PD1by, for example, co-localization (as assessed by, e.g.,immunohistochemistry) or co-immunoprecipitation (as assessed by, e.g.,western blot), in the presence of the negative control therapy.

In a specific embodiment, PD1 blockade refers to (i) inhibition ofligand-dependent PD1 activation, or (ii) blocking of ligand binding toPD1. In a specific embodiment, the inhibition or blocking is partial. Inanother specific embodiment, the inhibition or blocking is complete. Ina specific embodiment, PD1 blockade refers to least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% inhibition of ligand-dependent PD1 activation asassessed by any method known to one of skill in the art, such as, e.g.,a phosphorylation assay, as compared to ligand-dependent PD1 activationin the presence of a negative control therapy (e.g., an anti-IgGantibody). For example, in a specific embodiment, PD1 blockade that isinhibition of activation is assessed by (a) contacting a PD1ligand-expressing cell and a PD1-expressing activated T cell with ananti-PD1 therapy (e.g., an anti-PD1 antibody) or a negative controltherapy (e.g., an anti-IgG antibody), and (b) measuring thephosphorylation of PD1 or dephosphorylation of a downstream signalingmolecule, such as, e.g., Lck or Zap-70, as assessed by, for e.g., ELISAor western blot, in the presence of the anti-PD1 therapy as compared tothe phosphorylation of PD1 or dephosphorylation of a downstreamsignaling molecule, such as, e.g., Lck or Zap-70, as assessed by, fore.g., ELISA or western blot, in the presence of the negative controltherapy. In a specific embodiment, PD1 blockade that is blocking ofligand binding to PD1 is assessed by (a) contacting a ligand-expressingcell and a PD1-expressing activated T cell with an anti-PD1 therapy(e.g., an anti-PD1 antibody) or a negative control therapy (e.g., ananti-IgG antibody), and (b) measuring the interaction between ligand andPD1 by, for example, co-localization (as assessed by, e.g.,immunohistochemistry) or co-immunoprecipitation (as assessed by, e.g.,western blot) of ligand and PD1, in the presence of the anti-PD1 therapyas compared to the interaction between ligand and PD1 by, for example,co-localization (as assessed by, e.g., immunohistochemistry) orco-immunoprecipitation (as assessed by, e.g., western blot), in thepresence of the negative control therapy.

In a specific embodiment, an anti-PDL1 therapy is a PDL1-targetedtherapy that is effective in the treatment of one or more cancersexpressing PDL. In a specific embodiment, the anti-PDL1 therapycomprises an antibody or antigen-binding fragment thereof (e.g., a Fabfragment, a F(ab′)2 fragment, or a disulfide-linked Fv) orantigen-binding derivative thereof (e.g., a bispecific antibody, anscFv, an intrabody, or a camelized antibody), a polypeptide, aRNAi-inducing nucleic acid (e.g., an antisense oligonucleotide, a smallinterfering RNA, a microRNA, or a short hairpin RNA), or a smallmolecule that targets PDL1. Nonlimiting examples of an anti-PDL1 therapyinclude mpdl3280A (see, e.g., Herbst et al., J Clin Oncol. 2013;31(suppl):abstr 3000), durvalumab (e.g., for bladder cancer) (alsoreferred to as “medi-4736”; see, e.g., Lutzky et al., J Clin Oncol.2014; 32(suppl 5S):abstr 3001), avelumab (e.g., for Merkel cellcarcinoma) (also referred to as “MSB0010718C”; see, e.g., Heery et al. JClin Oncol. 2014; 32(suppl 5S):abstr 3064), and bms-936559 (see, e.g.,Brahmer et al. N. Engl. J. Med. 2012; 366, 2455-2465), and atezolizumab(see, e.g., McDermott et al., J Clin Oncol. 2016; 34(8):833-842). In apreferred embodiment, the anti-PDL1 therapy is an anti-PDL1 antibody. Ina preferred embodiment, the anti-PDL1 antibody is atezolizumab. In aspecific embodiment, the anti-PDL1 therapy is a therapy approved by theU.S. Food and Drug Administration (“FDA”) for treatment of one or morecancers. A nonlimiting example of an anti-PDL1 therapy approved by theU.S. Food and Drug Administration for treatment of cancer isatezolizumab. In a specific embodiment, the anti-PDL1 therapy is aPDL1-targeted therapy approved by the European Medicines Agency (“EMA”)for treatment of one or more cancers. A nonlimiting example of ananti-PDL1 therapy approved by the EMA for treatment of a PDL1-expressingcancer is atezolizumab.

In a specific embodiment, an anti-PD1 therapy is a PD1-targeted therapythat is effective in the treatment of one or more cancers expressingPDL1. In a specific embodiment, the anti-PD1 therapy comprises anantibody or antigen-binding fragment thereof (e.g., a Fab fragment, aF(ab′)2 fragment, or a disulfide-linked Fv) or antigen-bindingderivative thereof (e.g., a bispecific antibody, an scFv, an intrabody,or a camelized antibody), a polypeptide, a RNAi-inducing nucleic acid(e.g., an antisense oligonucleotide, a small interfering RNA, amicroRNA, or a short hairpin RNA), or a small molecule that targets PD1.Nonlimiting examples of an anti-PD1 therapy include nivolumab (see,e.g., Topalian et al., N Engl J Med. 2012; 366:2443-54), pidilizumab(see, e.g., Atkins et al., J Clin Oncol. 2014; 32(suppl 5S):abstr 9001),AMP-224 (see, e.g., Infante et al., J Clin Oncol. 2013; 31(suppl):abstr3044), MEDI0680 (also referred to as “AMP-514”; see, e.g., Hamid et al.,Ann Oncol. 2016; 27(suppl_6):1050PD), and pembrolizumab (see, e.g.,Hamid et al., N Engl J Med. 2013; 369:134-44). In a preferredembodiment, the anti-PD1 therapy is an anti-PD1 antibody. In a preferredembodiment, the anti-PD1 antibody is pembrolizumab. In a specificembodiment, the anti-PD1 therapy is a therapy approved by the U.S. FDAfor treatment of one or more cancers. Nonlimiting examples of ananti-PD1 therapy approved by the U.S. FDA for treatment of cancerinclude pembrolizumab and nivolumab. In a specific embodiment, theanti-PD1 therapy is a therapy approved by the EMA for treatment of oneor more cancers. Nonlimiting examples of an anti-PD1 therapy approved bythe EMA for treatment of cancer include pembrolizumab and nivolumab.

In contrast to trastuzumab (which is indicated for treatment ofHER2-overexpressing breast cancer, metastatic gastric cancer, andgastroesophageal junction adenocarcinoma (see Trastuzumab [Highlights ofPrescribing Information], South San Francisco, Calif.: Genentech, Inc.;2014)), the bispecific binding molecules described herein aretherapeutically effective against HER2-positive cancers that express lowlevels of HER2. See, e.g., the working example of Section 6.3, inparticular, FIG. 32, which demonstrates that tumor growth was completelysuppressed in a gastric cancer patient-derived xenograft model with lowHER2 expression when treated with a bispecific binding moleculedescribed herein; in contrast, treatment of the gastric cancerpatient-derived xenograft model with trastuzumab did not suppress tumorgrowth. Thus, also provided herein is a method of treating aHER2-positive cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of abispecific binding molecule as described in Section 5.1 or in Section5.2, a therapeutically effective amount of a cell, polynucleotide, orvector encoding such a bispecific binding molecule as described inSection 5.3, or a therapeutically effective amount of a pharmaceuticalcomposition as described in Section 5.5, or a therapeutically effectiveamount of T cells bound to a bispecific binding molecule as described inSection 5.4, wherein the cancer is not indicated for treatment withtrastuzumab, and wherein the cancer is not a head and neck cancer. In aspecific embodiment, the cancer is breast cancer. In a specificembodiment, the cancer is gastric cancer. In a specific embodiment, thecancer is gastroesophageal junction adenocarcinoma.

In a specific embodiment, the HER2-positive cancer is determined not tobe indicated for treatment with trastuzumab according to applicableAmerican Society of Clinical Oncology/College of American Pathologists(“ASCO/CAP”) guideline recommendations for HER2 testing in cancer (“ASCOHER2 Testing Guidelines”) (see, e.g., Wolff et al., Journal of ClinicalOncology, 2013, 31(31):3997-4013 and Bartley et al., Journal of ClinicalOncology, 2016, 146(6):647-669). The applicable ASCO HER2 TestingGuideline will be known to one of skill in the art. In a specificembodiment, the applicable ASCO HER2 Testing Guideline is the current(i.e., most recently published and updated) ASCO HER2 Testing Guidelineas of the date of using the ASCO HER2 Testing Guideline to determinethat the cancer is not indicated for treatment with trastuzumab. In analternative, preferred embodiment, the applicable ASCO HER2 TestingGuideline is the current (e.g., most recently published and updated)ASCO/CAP guideline recommendations for HER2 testing in breast cancer(“ASCO HER2 Breast Cancer Testing Guideline”) (see, e.g., Wolff et al.,Journal of Clinical Oncology, 2013, 31(31):3997-4013) as of the date ofdetermining that the cancer is not indicated for treatment withtrastuzumab, regardless of the type of cancer that is determined not tobe indicated for treatment with trastuzumab (e.g., the type of cancermay be breast cancer or any other HER2-positive cancer). In anotherembodiment, the applicable ASCO HER2 Testing Guideline is the current(e.g., most recently published and updated) ASCO HER2 Testing Guidelineas of the date of determining that the cancer is not indicated fortreatment with trastuzumab and the applicable ASCO HER2 TestingGuideline is for the same type of cancer (e.g., same tissue type, forexample, both being breast cancers, or both being gastric cancers) asthe cancer that is determined not to be indicated for treatment withtrastuzumab.

In a specific embodiment, the HER2-positive cancer is determined not tobe indicated for treatment with trastuzumab based on the followingcharacterization of the cancer (see, e.g., the 2013 ASCO HER2 BreastCancer Testing Guideline (e.g., as set forth in Wolff et al., Journal ofClinical Oncology, 2013, 31(31):3997-4013)): (a) a first determinationof a level of HER2 in a test specimen comprising cells of the cancer isreported as negative, or (b) a first determination of a level of HER2 ina test specimen comprising cells of the cancer is reported as equivocal,and a second determination of a level of HER2 in a test specimencomprising cells of the cancer is reported as equivocal or negative. Ina specific embodiment, the HER2-positive cancer is determined not to beindicated for treatment with trastuzumab when a first determination of alevel of HER2 in a test specimen comprising cells of the cancer isreported as negative according to the applicable ASCO HER2 TestingGuideline (e.g., the 2013 ASCO HER2 Breast Cancer Testing Guideline(e.g., as set forth in Wolff et al., Journal of Clinical Oncology, 2013,31(31):3997-4013)). In a specific embodiment, the HER2-positive canceris determined not to be indicated for treatment with trastuzumab when afirst determination of a level of HER2 in a test specimen comprisingcells of the cancer is reported as equivocal according to the applicableASCO HER2 Testing Guideline (e.g., the 2013 ASCO HER2 Breast CancerTesting Guideline (e.g., as set forth in Wolff et al., Journal ofClinical Oncology, 2013, 31(31):3997-4013)) and a second determinationof a level of HER2 in a test specimen comprising cells of the cancer isreported as equivocal or negative according to the applicable ASCO HER2Testing Guideline (e.g., the 2013 ASCO HER2 Breast Cancer TestingGuideline (e.g., as set forth in Wolff et al., Journal of ClinicalOncology, 2013, 31(31):3997-4013)). The test specimen can be from aprimary tumor or a metastatic tumor.

In a specific embodiment, the determination of the level of HER2 in thetest specimen is reported as negative when the level of HER2 in the testspecimen is characterized as (see, e.g., the 2013 ASCO HER2 BreastCancer Testing Guideline (e.g., as set forth in Wolff et al., Journal ofClinical Oncology, 2013, 31(31):3997-4013)): (i) (1) IHC 1+, wherein thelevel of HER2 in the test specimen is characterized as IHC 1+ when thetest specimen exhibits an incomplete HER2 membrane staining that isfaint/barely perceptible and within greater than 10% of the invasivetumor cells, wherein the staining is readily appreciated using alow-power objective; (2) IHC 0, wherein the level of HER2 in the testspecimen is characterized as IHC 0 when the test specimen exhibits nostaining observed, wherein the lack of staining is readily appreciatedusing a low-power objective, or a HER2 membrane staining that isincomplete and is faint/barely perceptible and within less than or equalto 10% of the invasive tumor cells, wherein the staining is readilyappreciated using a low-power objective; or (ii) ISH negative, whereinthe level of HER2 in the test specimen is characterized as ISH negativewhen the test specimen exhibits (1) a single-probe average HER2 copynumber of less than 4.0 signals per cell; or (2) a dual-probe HER2/CEP17ratio of less than 2.0 with an average HER2 copy number of less than 4.0signals per cell.

In a specific embodiment, the determination of the level of HER2 in thetest specimen is reported as equivocal when the level of HER2 in thetest specimen is characterized as (see, e.g., the 2013 ASCO HER2 BreastCancer Testing Guideline (e.g., as set forth in Wolff et al., Journal ofClinical Oncology, 2013, 31(31):3997-4013)): (i) IHC 2+, wherein thelevel of HER2 in the test specimen is characterized as IHC 2+ when thetest specimen exhibits (1) a circumferential HER2 membrane staining thatis incomplete and/or weak/moderate and within greater than 10% ofinvasive tumor cells, wherein the staining is observed in a homogenousand contiguous population, and wherein the staining is readilyappreciated using a low-power objective; or (2) a complete andcircumferential HER2 membrane staining that is intense and within lessthan or equal to 10% of invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; or (ii) ISH equivocal,wherein the level of HER2 in the test specimen is characterized as ISHequivocal when the test specimen exhibits which comprises: (1) asingle-probe ISH average HER2 copy number of greater than or equal to4.0 and less than 6.0 signals/cell, wherein the copy number isdetermined by counting at least 20 cells within the area and is observedin a homogenous and contiguous population; or (2) a dual-probeHER2/CEP17 ratio of less than 2.0 with an average HER2 copy number ofgreater than or equal to 4.0 and less than 6.0 signals per cell, whereinthe copy number is determined by counting at least 20 cells within thearea and is observed in a homogenous and contiguous population. In aspecific embodiment, when two determinations of the level of HER2 in asubject are made to determine that a cancer is not indicated fortreatment with trastuzumab (e.g., a first determination is reported asequivalent and a second determination is reported as equivalent ornegative), the two determinations are either: (1) based on the same testspecimen using different assays; or (2) based on different testspecimens using the same assay. For example, if the first determinationis based on a first test specimen using ISH, the second determination isbased on the first test specimen using IHC. In an alternative example,if the first determination is made based on a first test specimen usingISH, the second determination is based on a second test specimen usingISH.

In a specific embodiment, the level of HER2 in the test specimen isdetermined according to one or more assays approved by the U.S. Food andDrug Administration (“FDA”) for determining the level of HER2.Nonlimiting examples of U.S. Food and Drug Administration-approvedassays for determining a level of HER2 include HercepTest™ (manufacturedby DAKO), PATHWAY® (manufactured by Ventana Medical Systems Inc.),InSite® (manufactured by Biogenex Laboratories Inc.), Bond Oraclerm(manufactured by Leica Biosystems), PathVysion® (manufactured by AbbottMolecular Inc.), PharmDx™ Kit (manufactured by DAKO), SPoT-Light®(manufactured by Life Technologies Inc.), INFORM HER2 dual IDS DNA probecocktail (manufactured by Ventana Medical Systems Inc.), and PharmDx™(manufactured by DAKO). In another specific embodiment, the level ofHER2 in the test specimen is determined according to alaboratory-developed test performed in a Clinical Laboratory ImprovementAmendments-certified laboratory.

Also provided herein is a method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a bispecific binding molecule asdescribed in Section 5.1 or in Section 5.2, a therapeutically effectiveamount of a cell, polynucleotide, or vector encoding such a bispecificbinding molecule as described in Section 5.3, or a therapeuticallyeffective amount of a pharmaceutical composition as described in Section5.5, or a therapeutically effective amount of T cells bound to abispecific binding molecule as described in Section 5.4, wherein thecancer expresses a low level of HER2, and wherein the cancer is not ahead and neck cancer. In a specific embodiment, the cancer is breastcancer. In another specific embodiment, the cancer is gastric cancer. Ina specific embodiment, the cancer is gastroesophageal junctionadenocarcinoma.

In a preferred embodiment, the HER2-positive cancer is deemed to expressa low level of HER2 when a level of HER2 in a test specimen comprisingcells of the cancer is characterized as IHC 2+ or less (e.g., IHC 1+ orIHC 0) according to the applicable ASCO HER2 Testing Guideline. In aspecific embodiment, the HER2-positive cancer is deemed to express a lowlevel of HER2 when a level of HER2 in a test specimen comprising cellsof the cancer is characterized as IHC 2+ according to the applicableASCO HER2 Testing Guideline. In a specific embodiment, the HER2-positivecancer is deemed to express a low level of HER2 when a level of HER2 ina test specimen comprising cells of the cancer is characterized as IHC1+ according to the applicable ASCO HER2 Testing Guideline. In aspecific embodiment, the HER2-positive cancer is deemed to express a lowlevel of HER2 when a level of HER2 in a test specimen comprising cellsof the cancer is characterized as IHC 0 according to the applicable ASCOHER2 Testing Guideline. The applicable ASCO HER2 Testing Guideline willbe known to one of skill in the art. In a specific embodiment, theapplicable ASCO HER2 Testing Guideline is the current (e.g., mostrecently published and updated) ASCO HER2 Testing Guideline as of thedate of characterizing the level of HER2 in the test specimen comprisingcells of the cancer as IHC 2+ or less (e.g., IHC 1+ or IHC 0). In apreferred embodiment, the applicable ASCO HER2 Testing Guideline is thecurrent (e.g., most recently published and updated) ASCO HER2 BreastCancer Testing Guideline (see, e.g., Wolff et al., Journal of ClinicalOncology, 2013, 31(31):3997-4013) as of the date of characterizing thelevel of HER2 in the test specimen, regardless of the type of cancer ofthe test specimen (e.g., the test specimen may be of breast cancer orany other HER2-positive cancer). In another embodiment, the applicableASCO HER2 Testing Guideline is for the same type of cancer (e.g., sametissue type, for example, both being breast cancers, or both beinggastric cancers) as the cancer of the test specimen. In anotherembodiment, the applicable ASCO HER2 Testing Guideline is the current(e.g., most recently published and updated) ASCO HER2 Testing Guidelineas of the date of characterizing the level of HER2 in the test specimenand the applicable ASCO HER2 Testing Guideline is for the same type ofcancer (e.g., same tissue type, for example, both being breast cancers,or both being gastric cancers) as the test specimen. In a specificembodiment, the level of HER2 in the test specimen comprising cells ofthe cancer is characterized as IHC 2+ when the test specimen exhibits(see, e.g., Wolff et al., Journal of Clinical Oncology, 2013,31(31):3997-4013) (1) a circumferential HER2 membrane staining that isincomplete and/or weak/moderate and within greater than 10% of invasivetumor cells, wherein the staining is observed in a homogenous andcontiguous population, and wherein the staining is readily appreciatedusing a low-power objective; or (2) a complete and circumferential HER2membrane staining that is intense and within less than or equal to 10%of invasive tumor cells, wherein the staining is readily appreciatedusing a low-power objective. In a specific embodiment, the level of HER2in a test specimen comprising cells of the cancer is characterized asIHC 1+ when the test specimen exhibits (see, e.g., Wolff et al., Journalof Clinical Oncology, 2013, 31(31):3997-4013) an incomplete HER2membrane staining that is faint/barely perceptible and within greaterthan 10% of the invasive tumor cells, wherein the staining is readilyappreciated using a low-power objective. In a specific embodiment, thelevel of HER2 in the test specimen comprising cells of the cancer ischaracterized as IHC when the test specimen exhibits (see, e.g., Wolffet al., Journal of Clinical Oncology, 2013, 31(31):3997-4013) no HER2staining observed, wherein the lack of staining is readily appreciatedusing a low-power objective, or a HER2 membrane staining that isincomplete and is faint/barely perceptible and within less than or equalto 10%/6 of the invasive tumor cells, wherein the staining is readilyappreciated using a low-power objective.

In another embodiment, the HER2-positive cancer is deemed to express alow level of HER2 when the cancer expresses a lower level of HER2 thanthe level of HER2 expressed by cancers that are indicated for treatmentwith trastuzumab and are of the same type (e.g., same tissue type, forexample, both being breast cancers, or both being gastric cancers) asthe HER2-positive cancer. In a specific embodiment, the HER2 positivecancer is deemed to express a low level of HER2 when the HER2-positivecancer expresses a level of HER2 that is at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100/a lower than the level of HER2 expressedby cancers that are indicated for treatment with trastuzumab and are ofthe same type (e.g., same tissue type, for example, both being breastcancers, or both being gastric cancers) as the HER2-positive cancer. Ina specific embodiment, HER2 expression is measured by measuring HER2protein levels. In a specific embodiment, HER2 expression is measured bymeasuring HER2 nucleic acid levels (e.g, genomic DNA, cDNA, or RNAencoding HER2). In a specific embodiment, HER2 protein level is measuredaccording to any assay known in the art, such as, e.g., IHC, westernblot, enzyme-linked immunosorbent assay, or fluorescence-activated cellsorting. In a preferred embodiment, HER2 protein level is measuredaccording to IHC. In a specific embodiment, HER2 nucleic acid level ismeasured according to any assay known in the art, such as, e.g., ISH,southern blot, northern blot, quantitative reverse transcriptasepolymerase chain reaction, or deep sequencing. In a preferredembodiment, HER2 nucleic acid level is measured according to ISH.

In a specific embodiment, the level of HER2 in the test specimen isdetermined according to one or more assays approved by the U.S. Food andDrug Administration (“FDA”) for determining the level of HER2.Nonlimiting examples of U.S. Food and Drug Administration-approvedassays for determining a level of HER2 include HercepTest™ (manufacturedby DAKO), PATHWAY® (manufactured by Ventana Medical Systems Inc.),InSite® (manufactured by Biogenex Laboratories Inc.), Bond Oracle™(manufactured by Leica Biosystems), PathVysiong (manufactured by AbbottMolecular Inc.), PharmDx™ Kit (manufactured by DAKO), SPoT-Light®(manufactured by Life Technologies Inc.), INFORM HER2 dual IDS DNA probecocktail (manufactured by Ventana Medical Systems Inc.), and PharmDx™(manufactured by DAKO). In another specific embodiment, the level ofHER2 in the test specimen is determined according to alaboratory-developed test performed in a Clinical Laboratory ImprovementAmendments-certified laboratory.

In a specific embodiment, the HER2-positive cancer that expresses a lowlevel of HER2 is a breast cancer, a gastric cancer, an osteosarcoma,desmoplastic small round cell cancer, an ovarian cancer, a prostatecancer, a pancreatic cancer, glioblastoma multiforme, gastric junctionadenocarcinoma, gastroesophageal junction adenocarcinoma, a cervicalcancer, a salivary gland cancer, a soft tissue sarcoma, a leukemia, amelanoma, a Ewing's sarcoma, a rhabdomyosarcoma, a brain tumor, orneuroblastoma. In a preferred embodiment, the HER2-positive cancer thatexpresses a low level of HER2 is breast cancer. In a specificembodiment, the HER2-positive cancer that expresses a low level of HER2is gastric cancer. In a specific embodiment, the HER2-positive cancerthat expresses a low level of HER2 is ovarian cancer, pancreatic cancer,a desmoplastic small round cell tumor, an osteosarcoma, a melanoma, abrain tumor, a cervical cancer, a prostate cancer, or a salivary glandcancer. In a specific embodiment, the HER2-positive cancer thatexpresses a low level of HER2 is not a head and neck cancer.

In a specific embodiment, the HER2-positive cancer that expresses a lowlevel of HER2 is resistant to treatment with trastuzumab, cetuximab,lapatinib, erlotinib, or any other small molecule or antibody thattargets the HER family of receptors, and is responsive to treatment witha bispecific binding molecule of the invention. In a specificembodiment, the HER2-positive cancer that expresses a low level of HER2is resistant to treatment with necitumumab, pantitumumab, pertuzumab, orado-trastuzumab emtansine, and is responsive to treatment with abispecific binding molecule of the invention.

In a specific embodiment, a cancer is considered resistant to a therapy(e.g., an anti-PDL1 therapy, an anti-PD1 therapy, trastuzumab,cetuximab, necitumumab, panitumumab, pertuzumab, ado-trastuzumabemtansine, lapatinib, erlotinib, or any small molecule that targets theHER family of receptors) if it has no response, or has an incompleteresponse (a response that is less than a complete remission), orprogresses, or relapses after the therapy.

In specific embodiments, treatment can be to achieve beneficial ordesired clinical results including, but not limited to, alleviation of asymptom, diminishment of extent of a disease, stabilizing (i.e., notworsening) of state of a disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. In a specific embodiment, “treatment” can also be toprolong survival as compared to expected survival if not receivingtreatment.

5.6.2 Diagnostic Uses

In certain embodiments, bispecific binding molecules described hereincan be used for diagnostic purposes to detect, diagnose, or monitor acondition described herein (e.g., a condition involving HER2-positivecancer cells). In certain embodiments, bispecific binding molecules foruse in diagnostic purposes are labeled as described in Section 5.2.

In certain embodiments, provided herein are methods for the detection ofa condition described herein comprising (a) assaying the expression ofHER2 in cells or a tissue sample of a subject using one or morebispecific binding molecules described herein; and (b) comparing thelevel of HER2 expression with a control level, for example, levels innormal tissue samples (e.g., from a subject not having a conditiondescribed herein, or from the same patient before onset of thecondition), whereby an increase or decrease in the assayed level of HER2expression compared to the control level of HER2 expression isindicative of a condition described herein.

Antibodies described herein can be used to assay HER2 levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al.,1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In certain embodiments, monitoring of a condition described herein(e.g., a HER2-positive cancer), is carried out by repeating the methodfor diagnosing for a period of time after initial diagnosis.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. Skilled artisans will beable to determine the appropriate method for detecting a particularlabel. Methods and devices that may be used in the diagnostic methods ofthe invention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

5.7 Patient Population

A subject treated in accordance with the methods provided herein can beany mammal, such as a rodent, a cat, a canine, a horse, a cow, a pig, amonkey, a primate, or a human, etc. In a preferred embodiment, thesubject is a human. In another preferred embodiment, the subject is acanine.

In certain embodiments, a subject treated in accordance with the methodsprovided herein has been diagnosed with a HER2-positive cancer,including but not limited to, breast cancer, gastric cancer, anosteosarcoma, desmoplastic small round cell cancer, squamous cellcarcinoma of head and neck cancer, ovarian cancer, prostate cancer,pancreatic cancer, glioblastoma multiforme, gastric junctionadenocarcinoma, gastroesophageal junction adenocarcinoma, cervicalcancer, salivary gland cancer, soft tissue sarcoma, leukemia, melanoma,Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any otherneoplastic tissue that expresses the HER2 receptor.

In a specific embodiment, a subject treated in accordance with themethods provided herein has not been diagnosed with HER2-positivesquamous cell carcinoma of head and neck cancer.

In certain embodiments, the subject is resistant to treatment withtrastuzumab, cetuximab, lapatinib, erlotinib, or any other smallmolecule or antibody that targets the HER family of receptors. In aspecific embodiment, the tumor that is resistant to treatment withtrastuzumab, cetuximab, lapatinib, erlotinib, or any other smallmolecule or antibody that targets the HER family of receptors isresponsive to treatment with a bispecific binding molecule to theinvention.

In certain embodiments, a subject treated in accordance with the methodsprovided herein has a HER2-positive cancer that is resistant totreatment with trastuzumab, cetuximab, lapatinib, erlotinib, or anyother small molecule or antibody that targets the HER family ofreceptors. In certain embodiments, a subject treated in accordance withthe methods provided herein has a HER2-positive cancer that isresponsive to treatment with a bispecific binding molecule to theinvention.

In certain embodiments, the subject treated in accordance with themethods provided herein has previously received one or more chemotherapyregimens for metastatic disease, e.g., brain or peritoneal metastases.In certain embodiments, the subject has not previously receivedtreatment for metastatic disease.

5.8 Doses and Regimens

In certain embodiments, the dose of a bispecific binding molecule asdescribed in Section 5.1 administered to a subject according to themethods provided herein is a dose determined by the needs of thesubject. In certain embodiments, the dose is determined by a physicianaccording to the needs of the subject.

In a specific embodiment, the dose of a bispecific binding moleculeprovided herein is less than the dose of trastuzumab. See, for example,Trastuzumab [Highlights of Prescribing Information]. South SanFrancisco, Calif.: Genentech, Inc.; 2014. In a specific embodiment, thedose of a bispecific binding molecule provided herein is approximatelybetween 20 and 40 fold less than an FDA-approved dose of trastuzumab.

In certain embodiments, the dose of a bispecific binding molecule asdescribed in Section 5.1 administered to a subject according to themethods provided herein is between 0.01 mg/kg and 0.025 mg/kg, isbetween 0.025 mg/kg and 0.05 mg/kg, is between 0.05 mg/kg and 0.1 mg/kg,is between 0.1 mg/kg and 0.5 mg/kg, between 0.1 mg/kg and 0.6 mg/kg,between 0.2 mg/kg and 0.7 mg/kg, between 0.3 mg/kg and 0.8 mg/kg,between 0.4 mg/kg and 0.8 mg/kg, between 0.5 mg/kg and 0.9 mg/kg, orbetween 0.6 mg/kg and 1.

In certain embodiments, the dose of a bispecific binding molecule asdescribed in Section 5.1 administered to a subject according to themethods provided herein is an initial dose followed by an adjusted dosethat is the maintenance dose. In certain embodiments, the initial doseis administered once. In certain embodiments, the initial dose isbetween 0.01 mg/kg and 0.025 mg/kg, is between 0.025 mg/kg and 0.05mg/kg, is between 0.05 mg/kg and 0.1 mg/kg, is between 0.1 mg/kg and 0.5mg/kg, between 0.1 mg/kg and 0.6 mg/kg, between 0.2 mg/kg and 0.7 mg/kg,between 0.3 mg/kg and 0.8 mg/kg, between 0.4 mg/kg and 0.8 mg/kg,between 0.5 mg/kg and 0.9 mg/kg, or between 0.6 mg/kg and 1. In certainembodiments, the initial dose is administered via intravenous infusionover 90 minutes. In certain embodiments, the adjusted dose isadministered once every about 4 weeks. In certain embodiments, theadjusted dose is administered for at least 13, at least 26, or at most52 weeks. In certain embodiments, the adjusted dose is administered for52 weeks. In certain embodiments, the adjusted dose is between 0.01mg/kg and 0.025 mg/kg, is between 0.025 mg/kg and 0.05 mg/kg, is between0.05 mg/kg and 0.1 mg/kg, is between 0.1 mg/kg and 0.5 mg/kg, between0.1 mg/kg and 0.6 mg/kg, between 0.2 mg/kg and 0.7 mg/kg, between 0.3mg/kg and 0.8 mg/kg, between 0.4 mg/kg and 0.8 mg/kg, between 0.5 mg/kgand 0.9 mg/kg, or between 0.6 mg/kg and 1. In certain embodiments, theadjusted dose is administered via intravenous infusion over 30 minutes.In certain embodiments, the adjusted dose is administered viaintravenous infusion over 30 to 90 minutes.

In another specific embodiment, a bispecific binding molecule asdescribed in Section 5.1 for use with the methods provided herein isadministered 1, 2, or 3 times a week, every 1, 2, 3, or 4 weeks. Incertain embodiments, the bispecific binding molecule is administeredaccording to the following regimen: (1) 1, 2, or 3 administrations in afirst week; (ii) 1, 2, 3, or 4 administrations a week after the firstweek; followed by (iii) 1, 2, or 3 administrations in one week eachmonth for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Incertain embodiments, the bispecific binding molecule is administeredaccording to the following regimen: (i) 3 administrations in a firstweek; (ii) 3 administrations a week after the first week; followed by(iii) 3 administrations in one week each month for a total of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In certain embodiments, thebispecific binding molecule is administered according to the followingregimen: (i) 3 administrations in a first week; (ii) 2 administrations aweek after the first week; followed by (iii) 2 administrations in oneweek each month for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months. In certain embodiments, the bispecific binding molecule isadministered according to the following regimen: (i) 3 administrationsin a first week; (ii) 1 administrations a week after the first week;followed by (iii) 1 administrations in one week each month for a totalof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In certainembodiments, the bispecific binding molecule is administered accordingto the following regimen: (i) 2 administrations in a first week; (ii) 2administrations a week after the first week; followed by (iii) 2administrations in one week each month for a total of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 months. In certain embodiments, the bispecificbinding molecule is administered according to the following regimen: (i)2 administrations in a first week; (ii) 1 administrations a week afterthe first week; followed by (iii) 1 administrations in one week eachmonth for a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Incertain embodiments, the bispecific binding molecule is administeredaccording to the following regimen: (i) 1 administrations in a firstweek; (ii) 1 administrations a week after the first week; followed by(iii) 1 administrations in one week each month for a total of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In certain embodiments, a bispecific binding molecule as described inSection 5.1 is administered to a subject according to the methodsprovided herein in combination with a second pharmaceutically activeagent as described in Section 5.9.

In another preferred embodiment, the bispecific binding molecule isadministered intrathecally, intraventricularly in the brain,intraparenchymally in the brain, or intraperitoneally.

5.9 Combination Therapy

In certain embodiments, a bispecific binding molecule provided herein,or polynucleotide, vector, or cell encoding the bispecific bindingmolecule, may be administered in combination with one or more additionalpharmaceutically active agents, e.g., a cancer chemotherapeutic agent.In certain embodiments, such combination therapy may be achieved by wayof simultaneous, sequential, or separate dosing of the individualcomponents of the treatment. In certain embodiments, the bispecificbinding molecule or polynucleotide, vector, or cell encoding thebispecific binding molecule, and one or more additional pharmaceuticallyactive agents may be synergistic, such that the dose of either or ofboth of the components may be reduced as compared to the dose of eithercomponent that would be given as a monotherapy. Alternatively, Incertain embodiments, the bispecific binding molecule or polynucleotide,vector, or cell encoding the bispecific binding molecule and the one ormore additional pharmaceutically active agents may be additive, suchthat the dose of the bispecific binding molecule and of the one or moreadditional pharmaceutically active agents is similar or the same as thedose of either component that would be given as a monotherapy.

In certain embodiments, a bispecific binding molecule provided herein,or polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered on the same day as one or more additionalpharmaceutically active agents. In certain embodiments, the bispecificbinding molecule or polynucleotide, vector, or cell encoding thebispecific binding molecule is administered 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 hours before the one or more additional pharmaceuticallyactive agents. In certain embodiments, the bispecific binding moleculeor polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hoursafter the one or more additional pharmaceutically active agents. Incertain embodiments, the bispecific binding molecule or polynucleotide,vector, or cell encoding the bispecific binding molecule is administered1, 2, 3, or more days before the one or more additional pharmaceuticallyactive agents. In certain embodiments, the bispecific binding moleculeor polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered 1, 2, 3 or more days after the one or moreadditional pharmaceutically active agents. In certain embodiments, thebispecific binding molecule or polynucleotide, vector, or cell encodingthe bispecific binding molecule is administered 1, 2, 3, 4, 5, or 6weeks before the one or more additional pharmaceutically active agents.In certain embodiments, the bispecific binding molecule orpolynucleotide, vector, or cell encoding the bispecific binding moleculeis administered 1, 2, 3, 4, 5, or 6 weeks after the one or moreadditional pharmaceutically active agents.

In certain embodiments, the additional pharmaceutically active agent isdoxorubicin. In certain embodiments, the additional pharmaceuticallyactive agent is cyclophosphamide. In certain embodiments, the additionalpharmaceutically active agent is paclitaxel. In certain embodiments, theadditional pharmaceutically active agent is docetaxel. In certainembodiments, the one or more additional pharmaceutically active agentsis carboplatin.

In certain embodiments, the additional pharmaceutically active agent isa cytokine, such as, for example, IL15, IL15R/IL15 complex, IL2, orGMCSF.

In certain embodiments, the additional pharmaceutically active agent isan agent that increases cellular HER2 expression, such as, for example,external beam or radioimmunotherapy. See, for example, Wattenberg etal., 2014, British Journal of Cancer, 110: 1472.

In certain embodiments, the additional pharmaceutically active agent isa radiotherapeutic agent.

In certain embodiments, the additional pharmaceutically active agent isan agent that directly controls the HER2 signaling pathway, e.g.,lapatinib. See, for example, Scaltiri et al., 2012, 28(6): 803-814.

In certain embodiments, the additional pharmaceutically active agent isan agent that increases cell death, apoptosis, autophagy, or necrosis oftumor cells.

In certain embodiments, a bispecific binding molecule provided herein,or polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered in combination with two additionalpharmaceutically active agents, e.g., those used in combination withtrastuzumab (see, Trastuzumab [Highlights of Prescribing Information].South San Francisco, Calif.: Genentech, Inc.; 2014). In certainembodiments, the two additional pharmaceutically active agents aredoxorubicin and paclitaxel. In certain embodiments, the two additionalpharmaceutically active agents are doxorubicin and docetaxel. In certainembodiments, the two additional pharmaceutically active agents arecyclophosphamid and paclitaxel. In certain embodiments, the twoadditional pharmaceutically active agents are cyclophosphamide anddocetaxel. In certain embodiments, the two additional pharmaceuticallyactive agents are docetaxel and carboplatin. In certain embodiments, thetwo additional pharmaceutically active agents are cisplatin andcapecitabine. In certain embodiments, the two additionalpharmaceutically active agents are cisplatin and 5-fluorouracil.

In certain embodiments, a bispecific binding molecule provided herein,or polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered as a single agent following multi-modalityanthracycline based therapy.

In certain embodiments, a bispecific binding molecule provided herein,or polynucleotide, vector, or cell encoding the bispecific bindingmolecule is administered after one or more chemotherapy regimens formetastatic disease, e.g., brain or peritoneal metastases. In specificembodiments, a bispecific binding molecule provided herein, orpolynucleotide, vector, or cell encoding the bispecific binding moleculeis administered in combination with cytoreductive chemotherapy. In aspecific embodiment, the administering is performed after treating thesubject with cytoreductive chemotherapy.

In specific embodiments, a bispecific binding molecule provided herein,polynucleotide, vector, or cell encoding the bispecific bindingmolecule, or a pharmaceutical composition comprising the bispecificbinding molecule, is administered in combination with T cell infusion.In specific embodiments, the bispecific binding molecule is not bound toa T cell. In specific embodiments, the bispecific binding molecule isbound to a T cell. In specific embodiments, the binding of thebispecific binding molecule to the T cell is noncovalently. In aspecific embodiment, the administering of a bispecific binding moleculeprovided herein, polynucleotide, vector, or cell encoding the bispecificbinding molecule, or a pharmaceutical composition comprising thebispecific binding molecule is performed after treating the patient withT cell infusion. In specific embodiments the T cell infusion isperformed with T cells that are autologous to the subject to whom the Tcells are administered. In specific embodiments, the T cell infusion isperformed with T cells that are allogeneic to the subject to whom the Tcells are administered. In specific embodiments, the T cells can bebound to molecules identical to a bispecific binding molecule asdescribed herein. In specific embodiments, the binding of the T cells tomolecules identical to the bispecific binding molecule is noncovalently.In specific embodiments, the T cells are human T cells. Methods that canbe used to bind bispecific binding molecules to T cells are known in theart. &e, e.g., Lum et al., 2013, Biol Blood Marrow Transplant,19:925-33, Janeway et al., Immunobiology: The Immune System in Healthand Disease, 51^(th) edition, New York: Garland Science; Vaishampayan etal., 2015, Prostate Cancer, 2015:285193, and Stromnes et al., 2014,Immunol Rev. 257(1):145-164. See, also, Vaishampayan et al., 2015,Prostate Cancer, 2015:285193, which describes the following exemplary,non-limiting method for binding bispecific binding molecules to T cells:

-   -   Peripheral blood mononuclear cells (PBMCs) can be collected to        obtain lymphocytes for activated T cell expansion from 1 or 2        leukopheresis. PBMCs can be activated with, for example, 20        ng/mL of OKT3 and expanded in 100 IU/mL of IL-2 to generate        40-320 billion activated T cells during a maximum of 14 days of        culture under cGMP conditions as described in Ueda et al., 1993,        Transplantation, 56(2):351-356 and Uberti et al., 1994, Clinical        Immunology and Immunopathology, 70(3):234-240. Cells are grown        in breathable flasks (FEP Bag Type 750-Cl, American Fluoroseal        Corporation. Gaithersburg, Md.) in RPMI 1640 medium (Lonza)        supplemented with 2% pooled heat inactivated human serum.        Activated T cells are split approximately every 2-3 days based        on cell counts. After 14 days, activated T cells are cultured        with 50 ng of a bispecific binding molecule described herein per        10⁶ activated T cells. The mixture is then washed and        cryopreserved.

6. EXAMPLES 6.1 Example 1 6.1.1 Introduction

This example describes a HER2/CD3 bi-specific binding molecule (hereinreferred to as “HER2-BsAb”) based on an IgG1 platform. This platform wasutilized to allow for: (1) an optimal size to maximize tumor uptake, (2)bivalency towards the tumor target to maintain avidity, (3) a scaffoldthat is naturally assembled like any IgG (heavy chain and light chain)in CHO cells, purifiable by standard protein A affinity chromatography,(4) structural arrangement to render the anti-CD3 component functionallymonovalent, hence reducing spontaneous activation of T cells, and (5) aplatform with proven tumor targeting efficiency in animal models. Thisbispecific binding molecule has the same specificity as trastuzumab; butalso recruits and activates CD3(+) T cells redirecting them against HER2expressing tumor cells, generating robust anti-tumor responses. Withoutbeing bound by any theory, the effectiveness of this BsAb centers on theexploitation of the cytotoxic potential of polyclonal T cells, and itsunique capacity to target tumor cells that express even low levels ofHER2, independent of the activation status of the HER2 pathway.

6.1.2 Materials and Methods 6.1.2.1 HER2-BsAb Design, Production, andPurification Analyses

The HER2-BsAb format was designed as a huOKT3 scFv fusion to theC-terminus of the light chain of a human IgG1. The V_(H) was identicalto that of Trastuzumab IgG1, except N297A mutation in a standard humanIgG1 Fc region for aglycosylated form (SEQ ID NO: 62), while the lightchain is constructed as V_(L)-Cκ-(G₄S)₃-scFv (SEQ ID NO: 60). Nucleotidesequences encoding V_(H) and V_(L) domains from Trastuzumab, and thehuOKT3 scFv were synthesized by GenScript with appropriate flankingrestriction enzyme sites, and were subcloned into a standard mammalianexpression vector. HER2-C825 control BsAb (C825 is a murine scFvantibody with high affinity for1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-metalcomplexes with lanthanides including lutetium and yttrium) wasconstructed in a similar way.

Linearized plasmid DNA was used to transfect CHO-S cells (Invitrogen)for stable production of BsAb. 2×10⁶ cells were transfected with 5 μg ofplasmid DNA by Nucleofection (Lonza) and then recovered in CD OptiCHOmedium supplemented with 8 mM L-glutamine (Invitrogen) for 2 d at 37° C.in 6-well culture plates. Stable pools were selected with 500 μg/mLhygromycin for approximately two weeks and single clones were thenselected out with limited dilution. HER2-BsAb titer was determined byHER2(+) AU565 cell and CD3(+) Jurket cell ELISA, respectively, andstable clones with highest expression were selected.

The BsAb producer line was cultured in OptiCHO medium and the maturesupernatant harvested. A protein A affinity column (GE Healthcare) waspre-equilibrated with 25 mM sodium citrate buffer with 0.15 M NaCl, pH8.2. Bound BsAb was eluted with 0.1 M citric acid/sodium citrate buffer,pH 3.9 and neutralized with 25 mM sodium citrate, pH 8.5 (1:10 v/vratio). For storage, BsAb was dialyzed into 25 mM sodium citrate, 0.15 MNaCl, pH 8.2 and frozen in aliquots at −80° C. Two micrograms of theprotein was analyzed by SDS-PAGE under reducing conditions using 4-15%Tris-Glycine Ready Gel System (Bio-Rad). Invitrogen SeeBlue Plus2Pre-Stained Standard was used as the protein MW marker. Afterelectrophoresis, the gel was stained using Coomassie G-250 (GelCode BlueStain Reagent; Pierce). The purity of HER2-BsAb was also evaluated bysize-exclusion high-performance liquid chromatography (SE-HPLC).Approximately 20 μg of protein was injected into a TSK-GEL G3000SWXL 7.8mm×30 cm, 5 μm column (TOSOH Bioscience) with 0.4 M NaClO₄, 0.05 MNaH₂PO₄, pH 6.0 buffer at flow rate of 0.5 mL/min, and UV detection at280 nm. Ten microliters of gel-filtration standard (Bio-Rad) wasanalyzed in parallel for MW markers.

6.1.2.2 FACS Analyses

Cells were incubated with 5 μg/mL of primary antibody (trastuzumab,HER2-BsAb, or cetuximab) for thirty minutes at 4° C. in PBS, and asecondary phycoerythrin-labeled antibody specific for human Fc was usedafter wash of excess primary antibody. Cells were fixed with 1%paraformaldehyde (PFA) prior to analysis on FACSCalibur cytometer (BDbiosciences). Controls were cells with secondary antibody only, forwhich the mean fluorescent intensity (MFI) was set to 5. FACS datadisplay the MFI in the upper right panel of each plot.

6.1.2.3 ⁵¹Cr Release Assay

The ⁵¹Cr release assay was performed with effector T cells cultured invitro in the presence of anti-CD3 and anti-CD28 for about 14 days. Alltarget tumor cells were harvested with 2 mM EDTA in PBS, labeled with⁵¹Cr (Amersham, Arlington Height, Ill.) at 100 μCi/106 cells at 37° C.for 1 h. 5000 target cells/well were mixed with 50,000 effector cells(E:T=10:1) and BsAb antibodies in 96-well polystyrene round-bottomplates (BD Biosciences) to a final volume of 250 μl/well. The plateswere incubated at 37° C. for 4 h. The released ⁵¹Cr in supernatant wascounted in a γ-counter (Packed Instrument, Downers Grove, Ill.).Percentage of specific release was calculated using the formula:(experimental cpm−background cpm)/(total cpm−background cpm)×100%, wherecpm represented counts per minute of ⁵¹Cr released. Total release wasassessed by lysis with 10% SDS (Sigma, St Louis, Mo.), and backgroundrelease was measured in the absence of effector cells. EC50 wascalculated using SigmaPlot software.

6.1.2.4 Competition Assay

To assess the ability of trastuzumab and/or huOKT3 to interfere withHER2-BsAb binding, the HER2-positive SKOV3 cell line was incubated forthirty minutes a 4° C. with PBS or with 10 μg/mL of trastuzumab orhuOKT3. Cells were subsequently stained with 10 μg/mL of Alexa-Fluor488-conjugated HER2-BsAb and analyzed by flow cytometry. Alexa-Fluor488-conjugated HER2-BsAb was generated with the Zenon® Alexa Fluor® 488Human IgG Labeling Kit (Life Technologies) according to themanufacturer's instructions.

6.1.2.5 Binding Assay

Binding assays were performed by Surface Plasmon Resonance using BiacoreT100 similar as described in Okazaki et al., 2004, J Mol Biol; 336(5):1239-1249.

6.1.2.6 Avidity Assay

To compare the avidity of HER2-BsAb and trastuzumab, HER2-positive SKOV3cells were incubated with 10 fold dilutions (from 10 to 1×10⁻⁵ μg/mL) oftrastuzumab or HER2-BsAb and analyzed by flow cytometry withFITC-labeled human Fc-specific antibody as the secondary antibody. MFIwas plotted against the antibody concentration and the curves werecompared.

6.1.2.7 Proliferation Assay

To determine anti-proliferative effects, cells were treated with isotypecontrol monoclonal antibody, 10 nM lapatinib (as a positive control), 10μg/mL HER2-BsAb, 10 μg/mL Trastuzumab, 10 nM lapatinib, 10 nM erlotinib,10 nM neratinib, or 10 μg/mL cetuximab for 72 hours and cellproliferation assayed. Cell proliferation was determined using an ELISAplate reader and the WST-8 kit (Dojindo technologies) following themanufacturer's instructions and using the formula: % survivalrate=(Sample-Background)/(Negative control-Background). Lapatinib (MSKCCpharmacy) was ground using a mortar and pestle and suspended in DMSO aspreviously described. To determine statistical significance, the resultswere analyzed using one-way ANOVA using Prism 6.0.

6.1.2.8 qRT-PCR

RNA was extracted when cells were at 70% confluence and cDNA wasanalyzed in a prism 7700 sequence detection system using the HER2specific, commercially available kit Hs01001580_m1 from AppliedBiosciences.

6.1.2.9 Animals and In Vivo Assays

For in vivo studies, BALB-Rag2-KO-IL-2R-γc-KO (DKO) mice (derived fromcolony of Dr. Mamoru Ito, CIEA, Kawasaki, Japan). See, for example, Kooet al., 2009, Expert Rev Vaccines, 8: 113-120 and Andrade et al., 2011,Arthritis Rheum, 2011, 63: 2764-2773. MCF7 cells or HCC1954 were mixedat a 1:1 ratio with PMBCs (unactivated, from buffy coat) and implantedin DKO mice subcutaneously. Four days post implantation, mice weretreated with PBS, 10 μg of trastuzumab, or 10 μg of HER2-BsAb twice aweek for two weeks. Tumor size was measured at the indicated days postimplantation. Tumor size was determined by either calipers with theformula V=0.5 (length×width×width), or by using the Peira TM900 opticalsystem.

For the metastatic model, MCF7 cells expressing luciferase wereadministered to DKO mice intravenously. Four days post administration,mice were treated with 100 ug of HER2-BsAb, 20 ug or HER2-BsAb, or 20 ugof a HER2-BsAb lacking CD3 targeting (HER2-C825) twice a week for threeweeks, with or without intravenous administration of 5×10⁶ PBMC. Tumorsize was quantified at the indicated timepoints using IVIS 200 (Xenogen)to quantify luciferin bioluminescence.

6.1.3 Results 6.1.3.1 HER2-BsAb Binds to Both Tumor Cells and T Cells.

The HER2-BsAb was generated utilizing a trastuzumab variant comprising aN297A mutation in the human IgG1 Fc region to remove glycosylation (SEQID NO: 62). The BsAb light chain fusion polypeptide was generated byattaching the anti-CD3 humanized OKT3 (huOKT3) single chain Fv fragment(ScFv) to the carboxyl end of the trastuzumab IgG1 light chain via aC-terminal (G₄S)₃ linker (FIG. 1A and SEQ ID NO: 60). To avoidaggregation, a cysteine at position 105 of the variable heavy chain ofhuOKT3 was substituted with serine. A N297A mutation was also introducedinto the HER2-BsAb Fc region to eliminate binding of HER2-BsAb to Fcreceptors. This mutation has previously been shown to eliminate thecapacity of human IgG1-Fc binding to CD16A (FIG. 1D) and CD32A Fcreceptors (FIG. 1E).

To produce the HER2-BsAb, a mammalian expression vector encoding boththe heavy chain and the light chain fusion polypeptide was transfectedinto CHO-S cells, stable clones were selected, supernatants collected,and the HER2-BsAb was purified by protein A affinity chromatography.Biochemical purity analysis of the BsAb is depicted in FIG. 1B and FIG.1C. Under reducing SDS-PAGE conditions, HER2-BsAb gave rise to two bandsat around 50 KDa, since the huOKT3 scFv fusion to trastuzumab lightchain increased the MW to ˜50 KDa. SEC-HPLC showed a major peak (97% byUV analysis) with an approximate MW of 210 KDa, as well as a minor peakof multimers removable by gel filtration. The HER2-BsAb was stable bySDS-PAGE and SEC-HPLC after multiple freeze and thaw cycles.

FACS and immunostaining were performed to assess the binding ofHER2-BsAb to both target cells and effector cells. Trastuzumab andHER2-BsAb displayed comparable binding to the HER2-positive breastcarcinoma cell line, AU565 (FIG. 2A). In contrast, HER2-BsAbdemonstrated more than 20-fold less binding to CD3+ T cells than huOKT3(FIG. 2B). This is consistent with the observation that lightchain-anchored scFv had lower avidity for T cells than regular huOKT3IgG1, purposely designed to minimize cytokine release in the absence oftarget tumor cells.

The lower avidity of HER2-BsAb for T cells was further confirmed by thebinding affinity analysis by Biacore as described in Cheung et al. 2012,OncoImmunology, 1:477-486. For antigen CD3, HER2-BsAb had a k_(on) at4.53×10⁵ M⁻¹s⁻¹, a k_(off) at 8.68×10⁻² s⁻¹, and overall K_(D) at 192nM; comparable to parental huOKT3 IgG1-aGlyco at k_(off) (1.09×10⁻¹s⁻¹), but less at k_(on) (1.68×10⁶ M⁻¹s⁻¹) and overall Ki (64.6 nM). Insummary, HER2-BsAb had much lower k_(on) than its parentalhuOKT3-aGlyco, suggesting less chance of BsAb binding to and activatingT cells under the same condition, hence less cytokine release.

6.1.3.2 HER2-BsAb Redirected T Cell Killing of Human Tumor Cell Lines.

To evaluate whether HER2-BsAb could redirect T cells to kill tumorcells, T cell cytotoxicity on HER2(+) breast cancer AU565 cells wastested in a standard 4-hour ⁵¹Cr release assay. Substantial killing oftumor cells was observed n the presence of HER2-BsAb, with an EC50 at300 fM (FIG. 3). Moreover, the killing was effective for an extensivepanel of human tumor cell lines including breast carcinoma, ovariancarcinoma, melanoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma,and neuroblastoma, wherein the killing potency correlated with the HER2expression level in the cells by FACS (FIG. 4).

6.1.3.3 HER2-BsAb Mediates Tumor Antigen Specific T Cell Cytotoxicity.

To investigate the tumor antigen specificity of HER2-BsAb in T cellcytotoxicity, a cytotoxicity assay was performed in the HER2-positive UMSCC 47 cells (a model for head and neck cancer) and in the HER2-negativeHTB-132 cells (a model for breast cancer). HER2-BsAb mediated T cellcytotoxicity against the HER2-positive UM-SCC47 cells (EC50 of 14.5 μM),but not against the HER2-negative HTB-132 cells (FIG. 5A).

To investigate the specificity of HER2-BsAb in the T cell cytotoxicity,HER2-positive cells were first blocked with huOKT3 or with trastuzumab.In the absence of HER2-BsAb, the T cells displayed minimal cytotoxicity,reassuring that T cells on their own have minimum non-specificcytotoxicity. Both huOKT3 and trastuzumab blocked the ability ofHER2-BsAb to induce T cell cytotoxicity.

6.1.3.4 HER2-BsAb Mediates T Cell Cytotoxicity Against HER2-PositiveCells Below the HER2 Threshold of Detection by Flow Cytometry.

The HER2+ ovarian carcinoma cell line SKOV3 was used in a ⁵¹Crcytotoxicity assay with 10 fold dilutions of HER2-BsAb in the presenceof T cells. These same cells were stained using HER2-BsAb at the sameconcentrations and analyzed by flow cytometry, MFI was plotted over thesame x-axis as cytotoxicity, and EC50 was calculated for both curves.HER2-BsAb mediated T cell cytotoxicity against HER2-positive cells evenwhen HER2-BsAb binding was not detected by flow cytometry (FIG. 6).Comparing the EC50 for the cytotoxicity assay (2 μM) vs EC50 for flowcytometry curve (3.5 nM) suggests that T cells in the presence ofHER2-BsAb were 2500× more effective in detecting HER2-positive cellsthan flow cytometry.

6.1.3.5 HER2-BsAb has the Same Specificity, Affinity andAntiproliferative Effects as Trastuzumab.

Prior to treatment with HER2-BsAb, HER2-positive cells werepre-incubated with trastuzumab to determine if HER2-BsAb shares the sameantigen specificity as trastuzumab. Pre-incubation with trastuzumabblocked HER2-BsAb binding to the cells, demonstrating a sharedspecificity (FIG. 7A). To compare the affinity of HER2-BsAb totrastuzumab, HER2-positive cells were incubated with dilutions oftrastuzumab or HER2-BsAb and analyzed by flow cytometry for cellularbinding. Plotting of MFI against the antibody concentration revealedsimilar curves for trastuzumab and HER2-BsAb, demonstrating a similarbinding affinity (FIG. 7B). Further, trastuzumab and HER2-BsAbdemonstrated similar anti-proliferative effects against HER2-positivecells (FIG. 7C).

6.1.3.6 HER2-BsAb Mediated T Cell Cytotoxicity Against SCCHN with anEC50 in the Picomolar Range.

The level and frequency of HER2 in the previously characterized head andneck cancer cell lines 93-VU-147T, PCI-30, UD-SCC2, SCC90, UMSCC47 andPCI-15B were assessed via flow cytometry with trastuzumab. The cellswere also tested for HER2 expression by qRT-PCR (FIG. 8). HER2 wascomparably expressed in the panel of head and neck cancer cell lines.Finally, the level of cytotoxicity in the presence of T cells andHER2-BsAb was correlated with the level of HER2 in the cells, revealingHER2-BsAb displays an EC50 in the picomolar range for these head andneck cell lines (FIG. 8).

6.1.3.7 HER2-BsAb Mediates T Cell Cytotoxicity Against SCCHN Resistantto Other HER Targeted Therapies.

To determine the EGFR and HER2 status of the SCCHN cell line PCI-30,cells were stained with trastuzumab or cetuximab and analyzed by flowcytometry as previously described (FIG. 9A). A proliferation assaydemonstrated that these cells are resistant to the HER-specific targetedtherapies, trastuzumab, cetuximab, lapatinib, erlotinib and pan-HERinhibitor neratinib (FIG. 9B). However, PCI-30 cells were sensitive totreatment with HER2-BsAb utilizing three different cytotoxicity assays(FIG. 9C). HER2-BsAb generated potent cytotoxic responses against PCI-30independent of their sensitivity to other HER targeted therapies, evenwhen these drugs target more than one of these receptors. These assayssuggest that HER2-BsAb was able to generate powerful cytotoxicresponses, regardless of target cell sensitivity to EGFR or HER2targeted therapies.

6.1.3.8 HER2-BsAb Mediated T Cell Cytotoxicity Against Osteosarcoma CellLines with an EC50 in the Picomolar Range.

The previously characterized osteosarcoma cell lines, RG-160, CRL 1427and U2OS, were assessed for their HER2 expression by flow cytometry withtrastuzumab (FIG. 10) and by qRT-PCR, and the levels of HER2 werecorrelated with cytotoxicity in the presence of T cells and HER2-BsAb(FIG. 10). All tested cell lines were positive for HER2, although theexpression level varied. Further, all HER2-positive cells were sensitiveto T cell cytotoxicity mediated by HER2 BsAb, with an EC50 ranging from11-25 μM.

6.1.3.9 HER2-BsAb Mediates T Cell Cytotoxicity Against HER-TherapyResistant Osteosarcoma Cell Lines.

U2OS cells are a HER2-positive, EGFR-positive osteosarcoma cell line(FIG. 11A). U2OS cells were analyzed for their sensitivity totrastuzumab, cetuximab, lapatinib and the pan-HER inhibitor Neratinib byproliferation assay in the presence of each of the inhibitors. Thesecells were resistant to cetuximab and trastuzumab with minimalsensitivity to Lapatinib, erlotinib and neratinib (FIG. 11B). These samecells were tested for sensitivity for T cell cytotoxic responsesmediated by HER2-BsAb. HER2-BsAb generated potent cytotoxic responsesagainst U2OS cells using three different cytotoxicity assays,independent of its sensitivity to other HER targeted therapies (FIG.11C).

6.1.3.10 HER2-BsAb Mediates T Cell Cytotoxicity Against HER-TherapyResistant Cervical Cancer HeLa Cells.

HeLa cells are a HER2-positive, EGFR-positive cervical carcinoma cellline (FIG. 12A). HeLa cells were analyzed for their sensitivity to HERfamily tyrosine kinase inhibitors, Erlotinib, Lapatinib or Neratinib, orto the HER specific antibodies, Cetuximab or trastuzumab. These resultsdemonstrated that HeLa cells are pan-resistant to these therapies (FIG.12B). However, these same cells were tested for sensitivity for T cellcytotoxic responses mediated by HER2-BsAb. HER2-BsAb generated potentcytotoxic responses against HeLa cells using three differentcytotoxicity assays, independent of its sensitivity to other HERtargeted therapies (FIG. 12C). Interestingly, pretreatment withlapatinib increased sensitivity to HER2-BsAb mediated cytotoxicity, evenwhen lapatinib alone had no effect on cell proliferation.

6.1.3.11 HER2-BsAb is Effective Against Human Breast Cancer in HumanizedMice.

For in vivo therapy studies, BALB-Rag2-KO-IL-2R-γc-KO (DKO) mice(derived from colony of Dr. Mamoru Ito, CIEA, Kawasaki, Japan) wereused. See, for example, Koo et al. 2009, Expert Rev Vaccines 8: 113-120and Andrade et al. 2011, Arthritis Rhem 63: 2764-2773. MCF7-Luciferasebreast cancer cells were mixed with peripheral blood mononuclear cells(PBMC) and planted subcutaneously. Four days post cell implantation, themice were treated with HER2-BsAb or with trastuzumab and the tumor sizewas analyzed over time (FIG. 13). HER2-BsAb demonstrated a significantsuppression of tumor progression. HER2-BsAb was also effective againsttumor progression when the trastuzumab resistant HCC1954 breast cancercells (See, for example, Huang et al., 2011, Breast Cancer Research, 13:R84) were planted subcutaneously with PBMCs (FIG. 14).

To assess a metastatic tumor model, MCF7-Luciferace cells wereinoculated intravenously. HER2-BsAb was administered and subsequently incombination with PBMC. Tumor luciferin bioluminescence signaldemonstrated HER2-BsAb plus PBMC showed complete suppression of tumorprogression (FIG. 15, FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D).

6.1.4 Conclusions

The aglycosylated HER2-BsAb allowed for minimized Fc functions andavoidance of a cytokine storm and elimination of all complementactivation, complement mediated and complement receptor mediated immuneadherence. In addition, despite bivalency of huOKT3 in the IgG-scFvplatform, binding to CD3 was functionally monovalent; hence there was nospontaneous activation of T cells in the absence of tumor target.HER2-BsAb displayed potent cytotoxicity against HER2-positive tumorcells in vitro, even against cells with low antigen expression, or cellsthat are resistant to trastuzumab, cetuximab, lapatinib, erlotinib orthe pan-HER inhibitor neratinib. HER2-BsAb also displayed potentcytotoxicity against breast cancer, ovarian cancer, SCCHN,osteosarcomas, and sarcomas. Finally, HER2-BsAb displayed strong in vivoefficacy against tumor xenografts, substantially better than thetrastuzumab hIgG1 counterpart.

6.2 Example 2

This example provides (a) a more detailed description of certain of theexperiments described in Example 1 (Section 6.1); and (b) additionalexperiments as compared to Example 1 (Section 6.1).

6.2.1 Introduction

Trastuzumab has significantly improved patient outcomes in breast cancerand has also been key in the design and implementation of other targetedtherapies (Singh et al., 2014, Br J Cancer 111:1888-98). However, HER2expression does not guarantee a clinical response to trastuzumab orother HER2 targeted therapies (Gajria et al., 2011, Expert Review ofAnticancer Therapy, 11(2):263-75; Lipton et al., 2013, Breast CancerResearch and Treatment, 141(1):43-53). Less than 35% of patients withHER2 positive breast cancer initially respond to trastuzumab and 70% ofthe initial responders will ultimately progress with metastatic diseasewithin a year (Vu and Claret., 2011, Frontiers in Oncology 2:62). Inosteosarcoma and Ewing's sarcoma, where high levels of HER2 expressionare associated with decreased survival (Gorlick el al., 1999, Journal ofClinical Oncology: Official Journal of The American Society of ClinicalOncology 17:2781-2788), trastuzumab has not shown any benefit even whenused in conjunction with cytotoxic chemotherapy (Ebb et al., 2012,Journal of Clinical Oncology: Official Journal of the American Societyof Clinical Oncology 30:2545-2551). Furthermore, trastuzumab, like otherHER targeted therapies, has shown modest or no benefit againstHER2-positive head and neck cancer (Pollock et al., 2014, ClinicalCancer Research, 21(3):526-33).

The reasons for these failures are complex and only partiallyunderstood. The genomic diversity and constant evolution of malignanciesmake them less prone to oncogene addiction, a requirement for thesuccess of targeted therapy. Furthermore, even when oncogene addictionis present, resistance can emerge from selection pressure induced by theuse of targeted therapies (Lipton et al., 2013, Breast Cancer Researchand Treatment, 141(1):43-53). In fact, despite the initial enthusiasmreceived, the majority of targeted therapies have not produced asignificant benefit in the overall cure of patients receiving it(Nathanson et al., 2014, Science, 343:72-76). A different approach, onethat selectively targets malignant cells that overexpress HER familyreceptors, and that can generate cytotoxic anti-tumor responsesindependently of the receptor activation status can be beneficial.

Blinatumomab—a CD19/CD3 BsAb was approved in 2014 for treating AcuteLymphoplastic Leukemia (Sanford, 2015, Drugs 75:321-7). However, despiteits promising results, the unfavorable PK of these small size moleculesnecessitates prolonged infusions, complicating their administration(Shalaby et al., 1995, Clin Immunol Immunopathol 74:185-92, 1995;Portell et al., 2013, Clin Pharmacol 5:5-11). Furthermore, the resultingcytokine release syndrome (CRS) still poses costly and oftenlife-threatening complications. Importantly, despite the ability ofbispecific antibodies to activate T cells, the same inhibitory pathwaysthat regulate classic T cell function might still limit theireffectiveness. For example, the heterodimeric design of a monovalentbinding HER2/CD3 bispecific antibody was inhibited by the PD-1/PD-L1inhibitory axis (Junttila et al., 2014, Cancer Res 74:5561-71).

The present example provides a bispecific binding molecule (hereinreferred to as “HER2-BsAb”) that offers two distinct advantages over theexisting technologies: (1) it is based on the fully humanized HER2specific IgG1 mAb Trastuzumab, preserving its pharmacologic advantages(Wittrup et al., 2012, Methods Enzymol 503:255-68) and bivalent bindingto HER2; maximizing tumor avidity; and (2) its binding to CD3 isfunctionally monovalent through the scFv derived from the humanizedhuOKT3 mAb sequence. Thus, HER2-BsAb is built on two mAbs with extensiverecords of clinical safety. Furthermore, this is a platform with its Fcfunction deleted to eliminate all antibody-dependent cell-mediatedcytotoxicity (ADCC) and CMC activities in order to reduce the cytokinerelease syndrome.

The data presented in this example demonstrate the ability of HER2-BsAbto produce potent anti-tumor responses, both in vitro and in vivo,against tumor cells that are resistant to HER2 targeted therapy ortrastuzumab.

6.2.2 Materials and Methods 6.2.2.1 Cell Lines

All cell lines were purchased from ATCC (Manassas Va.) except: UM-SCC47,obtained from Dr. Carey at the University of Michigan; SCC-90, PCI-30and PCI-15B, obtained from Dr. Robert Ferris at the University ofPittsburgh; HCC1954, obtained from Dr. Sarat Chandarlapaty at MemorialSloan Kettering Cancer Center; 93-VU-147T and HeLa, obtained from Dr.Luc Morris; and UD-SCC2, obtained from Henning Bier atHals-Nasen-Ohrenklinik und Poliklinik. All cells were authenticated byshort tandem repeat profiling using PowerPlex 1.2 System (Promega), andperiodically tested for mycoplasma using a commercial kit (Lonza). Theluciferase-labeled tumor cell lines MCF7-Luc were generated byretroviral infection with a SFG-GFLuc vector.

6.2.2.2 HER2-BsAb Design and Expression in CHO-S Cells

In the HER2-BsAb IgG-scFv format (FIG. 17A, “HER2-BsAb”), the V_(H) wasidentical to that of the trastuzumab IgG1 V_(H), except that an N297Amutation in the Fc region was introduced into the HER2-BsAb to removeglycosylation, thereby depleting Fc function (SEQ ID NO: 62). The lightchain fusion polypeptide was constructed by extending the trastuzumabIgG1 light chain with a C-terminal (G₄S)₃ linker followed by huOKT3 scFv(SEQ ID NO: 60). The DNA encoding both the heavy chain and the lightchain was inserted into a mammalian expression vector, transfected intoCHO-S cells, and stable clones of the highest expression were selected.Supernatants were collected from shaker flasks and the HER2-BsAb waspurified by protein A affinity chromatography. The control BsAb,HER2-C825 (composed of SEQ ID NOS: 71 and 72), was generated aspreviously described (Xu et al., 2015, Cancer Immunol Res 3:266-77;Cheal et al., 2014, Mol Cancer Ther 13:1803-12).

6.2.2.3 Other Antibodies and Small Molecules

Fluorophore-labeled HER2-BsAb was generated with the Zenon® Alexa Fluor®488 Human IgG Labeling Kit from Life Technologies following themanufacturer's instructions. Pembrolizumab, cetuximab, trastuzumab,Erlotinib, Lapatinib and Neratinib were purchased from the MemorialSloan Kettering Cancer Center pharmacy. Small molecules werere-suspended in DMSO. The CD4, CD8, CD16 and CD56 antibodies werepurchased from BD Biosciences (San Jose Calif.). The commerciallyavailable PE labeled PD-L1 specific mAb 10F.9G2 was purchased fromBioLegend.

6.2.2.4 Cell Proliferation Assays

For cell proliferation assays, 5,000 tumor cells were plated usingRPMI-1640 supplemented with 10% FBS in a 96 well plate for 36 hoursbefore being treated with lapatinib or the antibodies at the specifiedconcentrations. Cell proliferation was determined using an ELISA platereader and the WST-8 kit (Dojindo technologies) following themanufacturer's instructions and using the formula: % survivalrate=(Sample-Background)/(Negative control-Background). Lapatinib(Memorial Sloan Kettering Cancer Center pharmacy) was ground using amortar and pestle and suspended in DMSO as previously described (Chen etal., 2012, Molecular cancer therapeutics 11:660-669). To determinestatistical significance, the results were analyzed using one-way ANOVAusing Prism 6.0.

6.2.2.5 Cytotoxicity Assays (⁵¹Chromium Release Assay)

Cell cytotoxicity was assayed by ⁵¹Cr release as previously described(Xu et al., 2015, Cancer Immunol Res 3:266-77), and EC50 was calculatedusing SigmaPlot software. Effector T cells were purified from human PBMCusing Pan T cell isolation kit (Miltenyi Biotec), and then activated andexpanded with CD3/CD28 Dynabeads (Invitrogen) according to themanufacturer's protocol.

6.2.2.6 PD-1/PD-L1 Expression

To overexpress PD-L1 in HEK293 cells, cells were cultured in DMEM(Celigro) supplemented with 10% heat-inactivated FBS and Penicillin (100IU/ml) and streptomycin (100 μg/ml). On Day(−1), HEK293 cells weretrypsinized, counted and plated into 6 well plates at 0.5 M cells/welland kept in 2 mL of fresh media. On the day of transfection, Day(0), themedia was exchanged with 2 mL of fresh media. Transfection reagents wereprepared as follows for both hPD-L1 and control plasmids: 2.5 μg of DNAwas diluted into 250 μl of unsupplemented DMEM (no serum). 5 μl ofLipofectamine 2000 (Invitrogen) was diluted into a separate 250 μl ofDMEM (no serum), and incubated for 5 minutes at room temperature. After5 minutes, the diluted DNA was combined with the diluted Lipofectamine2000 (Invitrogen) and incubated for another 30 minutes at roomtemperature. After 30 minutes, the entire 500 μl reaction was added,dropwise, onto a single well of HEK293 cells. The plate was rocked backand forth briefly to help mix the reagents. For the untransfectedcontrol, 500 μl of unsupplemented DMEM without DNA or Lipofectamine 2000was added to one well. Cells were incubated at 37° C. for 24-48 hoursbefore harvesting. On Day(1) or Day(2), cells were lifted from the plateusing 2 mM EDTA in PBS, and counted. 100,000-200,000 cells were used forFACS analysis and the rest were used for the killing assays.

To induce PD-1 expression of activated T cells (ATCs), effector cellswere incubated in a 3:1 ratio for 24 hours with the HER2-high BreastCarcinoma Cell line HCC1954 after these target cells were incubated withHER2-BsAb at a concentration of 10 μg/mL for 30 minutes and antibodyexcess was removed. Cells were harvested and used in cytotoxicity assaysas previously described against the HEK293 cells transfected with PD-L1.

6.2.2.7 In Vivo Experiments

For in vivo therapy studies, BALB-Rag2−/−IL-2R-γc-KO (“DKO”) mice(derived from colony of Dr. Mamoru Ito, CIEA, Kawasaki, Japan; see,e.g., Koo et al., 2009, Expert Rev Vaccines 8:113-20 and Andrade et al.,2011, Arthritis Rheum 63:2764-73) were used. Three humanized mousexenograft models were used: (1) intravenous tumor plus intravenouseffector cells; (2) subcutaneous tumor plus subcutaneous effector cells;and (3) subcutaneous tumor plus intravenous effector cells. Subcutaneousxenografts were created with 5×10⁶ cells suspended in Matrigel (CorningCorp, Tewksbury Mass.) and implanted in the flank of DKO mice. Effectorperipheral blood mononuclear cell (PBMC) cells were purified from buffycoats purchased from the New York Blood Center. Prior to everyexperimental procedure, PBMCs were analyzed for their percentage of CD3,CD4, CD8 and CD56 cells to ensure consistency. HER2-BsAb was injectedintravenously twice a week at 100 μg/injection, beginning two daysbefore effectors cells for three weeks, given as 5-10×10⁶ PBMC perinjection, once a week for 2 weeks. Tumor size was measured using (1)hand-held TM900 scanner (Pieira, Brussels, BE); (2) Calipers; or (3)bioluminescence. Bioluminescence imaging was conducted using the XenogenIn Vivo Imaging System (IVIS) 200 (Caliper LifeSciences). Briefly, micewere injected intravenously with 0.1 mL solution of D-luciferin (GoldBiotechnology; 30 mg/mL stock in PBS). Images were collected 1 to 2minutes after injection using the following parameters: a 10- to60-second exposure time, medium binning, and an 8 f/stop.Bioluminescence image analysis was performed using Living Image 2.6(Caliper LifeSciences).

6.2.3 Results 6.2.3.1 HER2-BsAb

HER2-BsAb was designed using an IgG-scFv format (FIG. 17A). The V_(H)was identical to that of trastuzumab IgG1, except for the N297A mutationin the Fc region of HER2-BsAb to remove glycosylation (SEQ ID NO: 62).The light chain fusion polypeptide was constructed by extending thetrastuzumab IgG1 light chain with a C-terminal (G₄S)₃ linker followed byhuOKT3 scFv (Xu et al., 2015, Cancer Immunol Res 3:266-77) (SEQ ID NO:60). The DNAs encoding both heavy chain and light chain were insertedinto a mammalian expression vector, transfected into CHO-S cells, andstable clones of highest expression were selected. Supernatants werecollected from shaker flasks and purified on protein A affinitychromatography.

SEC-HPLC and SDS-PAGE of the HER2-BsAb is shown in FIG. 17B and FIG.17C, respectively. Under reducing SDS-PAGE conditions, HER2-BsAb gaverise to two bands at around 50 kDa, since the huOKT3 scFv fusion totrastuzumab light chain increased the molecular weight to approximately50 kDa. SEC-HPLC showed a major peak (97% by UV analysis) with anapproximate molecular weight of 200 KDa, as well as a minor peak ofmultimers removable by gel filtration. The BsAb remained stable bySDS-PAGE and SEC-HPLC after multiple freeze and thaw cycles.

6.2.3.2 HER2-BsAb Retained Specificity, Affinity and Anti-ProliferativeEffects of Trastuzumab

To determine if HER2-BsAb retained the specificity andanti-proliferative effects of trastuzumab, the HER2-positive-high SKOV3ovarian carcinoma cell line was pre-incubated with 10 μg/mL oftrastuzumab for 30 minutes and then immunostained using HER2-BsAblabeled with Alexa 488 (FIG. 18A). Incubation with trastuzumab preventedHER2-BsAb binding to SKOV3 cells, demonstrating that these antibodiesshared the same specificity. To compare the avidity of HER2-BsAb totrastuzumab, the same cell line was incubated with 10-fold downwarddilutions (from 10 μg/ml to 1×10⁻⁵ μg/mL) of trastuzumab or HER2-BsAband analyzed by flow cytometry. The mean fluorescence intensity (MFI)was plotted against the antibody concentration in μM. The similarity inthe binding curves confirmed that trastuzumab and HER2-BsAb had similarbinding avidities for their common HER2 target (FIG. 18B).

Finally, the trastuzumab-sensitive breast carcinoma cell line SKBR3 wastreated with isotype control mAb, 10 mM Lapatinib (as a positivecontrol), 10 μg/mL HER2-BsAb, or 10 μg/mL trastuzumab for 72 hours andcell proliferation was assayed. As shown in FIG. 18C, trastuzumab andHER2-BsAb had similar anti-proliferative effects that were significantas compared to the negative control. As expected, lapatinib showed thestrongest inhibition of cell proliferation.

6.2.3.3 HER2-BsAb Redirected T Cell Cytotoxicity was HER2-Specific andDependent on CD3

To establish the specificity of cytotoxic responses by T cells in thepresence of HER2-BsAb; HER2-negative and HER2-positive cell lines wereassayed in a cytotoxicity assays using ATCs (effector:T cell (“E:T”)ratio of 10:1) and HER2-BsAb at decreasing concentrations (FIG. 19A andFIG. 20). Cytotoxicity was absent for HER2-negative cell lines. Todemonstrate the dependency of cytotoxicity on CD3, HER2-BsAbcytotoxicity was tested in the presence of the CD3 specific blocking mAbOKT3 (FIG. 19B). Pre-incubation with either trastuzumab or OKT3prevented HER2-BsAb T cell mediated cytotoxicity.

6.2.3.4 HER2-BsAb Mediated Cytotoxicity Against HER2-Positive Cell Linesthat were Resistant to Other HER2 Targeted Therapies.

Several cell lines from different tumor systems (e.g., head and neck,breast, and sarcoma) were characterized for their HER2 level ofexpression by flow cytometry (FIG. 20). In this panel, 75% of thesecells tested positive for HER2 expression by flow cytometry.Representative cell lines were assayed for their sensitivity to tyrosinekinase inhibitors (e.g., erlotinib, lapatinib, and neratinib), or HERantibodies (e.g., trastuzumab and cetuximab), as well as HER2-BsAbmediated T cell cytotoxicity. FIG. 21 shows representative examples ofthese experiments from three different lines from three different tumorsystems. As shown, HER2 expression-even in low quantities—was sufficientto mediate T cell cytotoxicity in the presence of ATC and HER2-BsAb incell lines otherwise resistant in vitro to HER-targeted therapies. Whenthese cell lines were tested for cytotoxicity in the presence of ATC andHER2-BsAb, sensitivity to HER2-BsAb, expressed as EC50, stronglycorrelated with surface HER2 expression (FIG. 22)

6.2.3.5 HER2-BsAb Mediated T Cell Cytotoxicity was RelativelyInsensitive to PD-L1 Expression on the Tumor Target or PD-1 Expressionon T Cells.

Activation of tumor-specific CTL in the tumor microenvironment is knownto promote expression of PD-1/PD-L1, leading to T cell exhaustion orsuppression, a phenomenon termed “adaptive immune resistance” (Tumeh etal., 2014, Nature 515:568-71). The presence of the PD-1/PD-L1 pathwayhas also been reported to limit the anti-tumor effects of T cellengaging bispecific antibodies (Junttila et al., 2014, Cancer Res74:5561-71). To determine if HER2-BsAb had the same limitations,PD-1-positive ATCs were used against the HER2-positive, PD-L1-positivebreast carcinoma cell line HCC1954, with or without the PD-1-specificmAb pembrolizumab. As shown in FIG. 23A, FIG. 23B, and FIG. 23C,PD-1-positive T cells generated similar cytotoxic responses in thepresence of HER2-BsAb, independently of the presence of pembrolizumab.When HER2-positive human embryonic kidney cells (HEK-293) weretransfected with the full sequence of PD-L1 and used as targets,cytotoxicity against cells expressing PD-L1 was not significantlydifferent to the cytotoxicity observed in non-transfected HEK-293 cells(although maximal cytotoxicity was slightly less with PD-L1-positiveHEK-293 versus PD-L1-negative HEK-293) (FIG. 24A and FIG. 24B shows theaverage of six experiments, and error bars represent standard error).

6.2.3.6 HER2-BsAb was Effective Against HER2-Positive Xenografts

To determine the in vivo efficacy of HER2-BsAb, the breast carcinomacell lines HCC1954 (HER2-high) and MCF-7 (HER2-low) were used inxenograft models in DKO mice. Three tumor models differing in tumorlocations and effector routes were used: (1) intravenous tumor cells andintravenous effector PBMCs; (2) subcutaneous tumor cells and SC PBMCs;and (3) subcutaneous tumor cells and intravenous PBMCs. FIG. 25summarizes the results of these experiments. The HER2-low MCF-7-luc(carrying luciferase reporter) cells were inoculated via tail veininjection into DKO. When tumor presence was confirmed bybioluminescence, mice were treated with six doses of intravenousHER2-BsAb or control BsAb twice a week for 3 weeks. Intravenous effectorPBMCs were administered 48 hours after the first dose of HER2-BsAb, andagain (one week later). Mice were evaluated for tumor burden usingluciferin bioluminescence every week. In this hematogenous diseasemodel, MCF-7 cells were completely eradicated without diseaseprogression (FIG. 25B). This same cell line was implanted subcutaneouslymixed with effector PBMCs subcutaneously and treated with fourinjections of HER2-BsAb twice a week for 2 weeks (totaling 4 injectionsin the first experiment) or twice a week for 3 weeks (totaling 6injections in 2nd experiment). In both experiments, HER2-BsAb caused asignificant delay in tumor progression while PBMC+trastuzumab or PBMCalone were ineffective (FIG. 25A). In two other separate experiments,subcutaneous HER2-positive breast carcinoma cell line HCC1954 was mixedwith subcutaneous PBMCs. Again, both 4 or 6 injections of HER2-BsAbresulted in a complete suppression of tumor growth, while trastuzumab orcontrol BsAb HER2-C825 had no effect (FIG. 25C). In the third model,where subcutaneous HCC1954 xenografts were treated with intravenous PBMC(once a week for 3 weeks), and intravenous HER2-BsAb twice a week for 3weeks, tumor growth was substantially delayed (in 2 separateexperiments), in contrast to only modest effects fortrastuzumab+huOKT3+PBMC, control antibody (HER2-C825)+PBMC, huOKT3+PBMC,or HER2-BsAb alone without PBMC (FIG. 25D). The following observationwere made: when effector PBMCs were mixed with tumor cellssubcutaneously, complete tumor regression without recurrence was seenfor mice over 90 days post-tumor implantation. When effector PBMCs wereadministered intravenously, there was significant reduction in the sizeof the tumors, but complete regression was only observed in a subset ofanimals.

6.2.4 Conclusions

This example describes a HER2-specific BsAb that has been shown to havepotent T cell-mediated anti-tumor activity in vitro and in vivo,ablating tumors or delaying tumor growth in 3 separate tumor models inthe presence of human PBMCs. Unlike monovalent bispecific antibodies,this HER2-BsAb had identical anti-proliferative capacity as trastuzumab.In addition, the serum half-life and area under the curve of HER2-BsAbwere similar to IgG. Unlike other bispecific antibodies, which tended toaggregate, HER2-BsAb was stable at −20° C. and at 37° C., despite longterm storage. Most importantly, the T cell-mediated cytotoxicity itinduced was relatively insensitive to inhibition by the PD-1/PD-L1pathway.

When compared to the existing platforms that target HER2, HER2-BsAboffers advantages. The F(ab)×F(ab) format, though effective in vitro,was similar in size to Blinatumomab (Sanford, 2015, Drugs, 75:321-7) andwas expected to share similar pharmacokinetic and toxicity profiles(Shalaby et al., 1995, Clin Immunol Immunopathol 74:185-92, 1995),having a short half-life, thus requiring daily infusions, potentialleakage into the central nervous system (CNS), potential CNS toxicity,and potential significant cytokine release syndrome. In addition, theanti-proliferative capacity of this F(ab)×F(ab) univalent system was10-fold lower than trastuzumab. The IgG×IgG chemical conjugate betweentrastuzumab and OKT3 was useful for arming T cells ex vivo, but was notuseful as an injectable, likely due to impurities associated withchemical conjugates (Lum and Thakur, 2011, BioDrugs 25:365-79; Lum etal., Clin Cancer Res 21:2305, 2015); in contrast, the HER2-BsAb providedherein is tolerated as an injectable. A heterodimer format was recentlydescribed using a monovalent system (Junttila et al., 2014, Cancer Res74:5561-71) that does not preserve trastuzumab's anti-proliferativeeffects retained in HER2-BsAb.

There are other design features that distinguish HER2-BsAb from otherknown candidates of this class. Unlike most bispecific antibodies,HER2-BsAb's bivalent binding to the HER2 target was preserved, providinganti-proliferative activity similar to that of trastuzumab IgG1. UnlikeF(ab)×F(ab) (Shalaby et al., 1995, Clin Immunol Immunopathol 74:185-92)or tandem scFv constructs (Sanford, 2015, Drugs, 75:321-7), HER2-BsAbhad a molecular weight high enough to behave in pharmacokinetic analyseslike a wild-type IgG. Unlike other bivalent bispecifics (Reusch et al.,MAbs, 7:584, 2015), HER2-BsAb's reaction with CD3 was functionallymonovalent. HER2-BsAb also differed from man heterodimeric bispecificsin its modified Fc, where aglycosylation removed both ADCC and CMCfunctions, thereby reducing cytokine release syndrome without affectingserum pharmacokinetics or compromising T cell activation. The otheradvantage is manufacturability; HER2-BsAb was produced in CHO cells andpurified using procedures standard for IgG, without significantaggregation despite prolonged incubation at 37° C. HER2-BsAb is animportant salvage option for patients who progress on standardHER2-based therapies, or a replacement for trastuzumab given its dualanti-proliferative and T cell retargeting properties.

6.3 Example 3

This example provides (a) a more detailed description of certain of theexperiments described in Example 1 (Section 6.1); and (b) additionalexperiments as compared to Example 1 (Section 6.1).

T-cell based therapies have emerged as one of the most clinicallyeffective ways to target solid and non-solid tumors. HER2 is responsiblefor the oncogenesis and treatment resistance of several human solidtumors. As a member of the HER family of tyrosine kinase receptors, itsover-activity confers unfavorable clinical outcome. Targeted therapiesdirected at this receptor have achieved responses, although developmentof resistance is common. This example explores a novel HER2/CD3bispecific antibody (HER2-BsAb) platform that, while preserving theanti-proliferative effects of trastuzumab, recruits and activatesnon-specific circulating T-cells, promoting T cell tumor infiltrationand ablating HER2-positive (“HER2(+)”) tumors, even when these areresistant to standard HER2 targeted therapies. Its in vitro tumorcytotoxicity, when expressed as EC50, correlated with the surface HER2expression in a large panel of human tumor cell lines, irrespective oflineage or tumor type. HER2-BsAb-mediated cytotoxicity was relativelyinsensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separatehumanized mouse models of human breast cancer and ovarian cancer cellline xenografts, as well as in human breast cancer and gastric cancerpatient-derived xenografts (“PDXs”), HER2-BsAb was highly effective inpromoting T cell infiltration and suppressing tumor growth when used inthe presence of human peripheral blood mononuclear cells (“PBMC”) oractivated T cells (“ATC”). The in vivo and in vitro antitumor propertiesof this BsAb support its further clinical development as a cancerimmunotherapeutic.

6.3.1 Introduction

Trastuzumab has significantly improved patient outcomes in breastcancer, and has also been key in the design and implementation of othertargeted therapies (Singh et al., Br J Cancer 2014; 111:1888-98).However, HER2 expression does not guarantee a clinical response totrastuzumab or other HER2 targeted therapies (Devika & Sarat, ExpertReview Of Anticancer Therapy 2011; 11(2):263-75; Lipton et al., BreastCancer Research and Treatment 2013; 141(1):43-53). HER2-positive breastcancer patients with metastatic disease initially respond to trastuzumaband/or other HER2 targeted therapies, but almost all eventually willdevelop resistance and relapse (Montemurro & Scaltriti, J Pathol 2014;232:219-29). In osteosarcoma and Ewing's sarcoma, where high levels ofHER2 expression was associated with decreased survival (Gorlick et al.,J Clin Oncol 1999; 17:2781-8), trastuzumab has not shown any benefiteven when used in conjunction with cytotoxic chemotherapy (Ebb et al., JClin Oncol 2012; 30:2545-51). Furthermore, trastuzumab, like other HERtargeted therapies, has shown modest or no benefit against HER2(+)positive head and neck cancer (Pollock & Grandis, Clinical CancerResearch 2014; 21(3):526-33).

The reasons for these failures are complex and only partiallyunderstood. The genomic diversity and constant evolution of malignanciesmake them less prone to oncogene addiction, a requirement for thesuccess of targeted therapy. Furthermore, even when oncogene addictionis present, resistance can emerge from selection pressure induced by theuse of targeted therapies (Lipton et al., Breast Cancer Research andTreatment 2013; 141(1):43-53). In fact, despite the initial enthusiasmreceived, the majority of targeted therapies have not produced asignificant benefit in the overall cure of patients receiving them(Nathanson et al., Science 2014; 343:72-6). A different approach, onethat selectively targets malignant cells that overexpress HER familyreceptors, and that can generate cytotoxic anti-tumor responsesindependently of the receptor activation status could be beneficial.

Redirecting the immune system against tumor cells has gained acceptanceas an effective strategy to overcome resistance to cytotoxicchemotherapy and targeted therapy. In the forefront of these treatments,T-cell based therapies constitute the most promising approach. BothT-cell engaging bispecific antibodies and immune checkpoint antibodyblockade have received accelerated approval from the FDA based on theiroutstanding clinical performance (Asher, Nature Reviews Drug Discovery2015). The clinical success of chimeric antigen receptor (CAR) genemodified T-cells against non-solid tumors has further added to theenthusiasm among scientists, clinicians and the pharmaceutical industry.

The outstanding clinical responses seen with these therapies haveconsolidated T-cells as the most powerful effector cells within theimmune system to eradicate tumor cells (Kershew et al., Clinical &Translational Immunology 2014; 3(5):e16). Thus, a number of approachesthat redirect them against tumor cells have been proposed and tested bymany investigators. In this regard, Bispecific antibodies (“BsAb”) withspecificity for T-cells and for tumor antigens have attracted theattention of researchers and big pharma. BsAb, in opposition to otherantibody based therapies, only requires expression of its target ofinterest to be effective. By recruiting polyclonal T-cells through theCD3 surface receptor, BsAb activate T-cells irrespective of theirlineage, antigen specificity, maturation, HLA restriction orco-stimulatory receptors. The direct activation of T-cells, bypassingthe classic T cell receptor (“TCR”), removes the limitations imposed byHLA restriction and its level of expression (Brischwein et al., Journalof Immunotherapy 2006; 30:798-807), a well-established immune resistancemechanism (Sabbatino et al., Clinical transplants 2013:453-63).

Blinatumomab—a CD19/CD3 BsAb was approved in 2014 for treating AcuteLymphoplastic Leukemia (Sanford, Drugs 2015; 75:321-7). However, despiteits promising results, the unfavorable pharmacokinetics of these smallsize molecules necessitate prolonged infusions, complicating theiradministration (Shalaby et al., J Exp Med 1992; 175:217-25; Portell etal., Clinical Pharmacology: Advances And Applications 2013; 5:5-11).Furthermore, the resulting cytokine release syndrome (“CRS”) still posescostly and often life-threatening complications. Importantly, despitethe ability of bispecific antibodies to activate T-cells, the sameinhibitory pathways that regulate classic T-cell function might stilllimit their effectiveness. For example, the heterodimeric design of amonovalent binding HER2/CD3 bispecific antibody was inhibited by thePD-1/PD-L1 inhibitory axis (Junttila et al., CancerResearch 2014;74:5561-71).

This example reports a BsAb against the HER2 tumor antigen that offerstwo distinct advantages over the existing technologies: (1) it is basedon the fully humanized HER2-specific IgG1 trastuzumab, preserving itspharmacologic advantages (Wittrup et al., Methods Enzymol 2012;503:255-68) and bivalent binding to HER2, maximizing tumor avidity; (2)its binding to CD3 is functionally monovalent through the scFv derivedfrom the humanized huOKT3 IgG1 sequence. Thus, HER2-BsAb is built on twomAbs with an extensive record of clinical safety. Previous studies havealso shown that scFv linked to the carboxyl end of the light chain didnot affect the targeting ability of these IgG forms (Cheal et al., MolCancer Ther 2014; 13:1803-12; Orcutt et al., Protein Eng Des Sel 2010;23:221-8). Furthermore, this is a platform with its Fc function silencedto reduce the cytokine release syndrome. This example presents data toshow that this HER2-BsAb has potent anti-tumor properties both in vitroand in vivo, against tumor cells that are resistant to HER2 targetedtherapy or to trastuzumab.

6.3.2 Materials and Methods 6.3.2.1 Cell Lines

All cell lines were purchased from ATCC (Manassas Va.) except: UM-SCC47obtained from Dr. Carey at the University of Michigan, SCC-90, PCI-30and PCI-15B from Dr. Robert Ferris at the University of Pittsburgh,SKOV3-luc from Dr. Dmitry Pankov at MSK, 93-VU-147T and HeLa from Dr.Luc Morris and UD-SCC2 from Henning Bier at Hals-Nasen-Ohrenklinik undPoliklinik. All cells were authenticated by short tandem repeatprofiling using PowerPlex 1.2 System (Promega), and periodically testedfor mycoplasma using a commercial kit (Lonza). The luciferase-labeledtumor cell lines MCF7-Luc were generated by retroviral infection with aSFG-GFLuc vector.

6.3.2.2 HER2-BsAb Design and Expression in CHO-S Cells

In the HER2-BsAb IgG-scFv format, VH was identical to that oftrastuzumab IgG1, except N297A mutation in the Fc region was introducedto remove glycosylation, thereby depleting Fc function. The sequence ofthe heavy chain is set forth in SEQ ID NO: 62. The light chain fusionpolypeptide (SEQ ID NO: 60) was constructed by extending the trastuzumabIgG1 light chain with a C-terminal (G₄S)₃ linker followed by huOKT3scFv. The DNA encoding both heavy chain and light chain was insertedinto a mammalian expression vector, transfected into CHO-S cells, andstable clones of highest expression were selected. Supernatants werecollected from shaker flasks and the HER2-BsAb was purified by protein Aaffinity chromatography. The other control BsAb, HER2-C825, wasgenerated as previously described (Cheal et al., Mol Cancer Ther 2014;13:1803-12). HuOKT3 IgG1 was made using the same variable sequences asin huOKT3 scFv, and huOKT3 Fab was prepared from huOKT3 IgG1 using thePierce Fab Preparation Kit (Thermo Scientific).

6.3.2.3 Other Antibodies and Small Molecules

Fluorophore-labeled HER2-BsAb was generated with the Zenon® Alexa Fluor®488 Human IgG Labeling Kit from Life Technologies following themanufacturer's instructions. Pembrolizumab, cetuximab, trastuzumab,Erlotinib, Lapatinib and Neratinib were purchased from the MemorialSloan Kettering Cancer Center pharmacy. Small molecules werere-suspended in dimethylsulfoxide (“DMSO”). The CD3, CD4, CD8 and CD56antibodies were purchased from BD Biosciences (San Jose Calif.). Thecommercially available PE labeled PD-L1 specific mAb 10F.9G2 waspurchased from BioLegend.

6.3.2.4 Cell Proliferation Assays

For tumor cell proliferation, 5,000 tumor cells were plated usingRPMI-1640 supplemented with 10%, fetal bovine serum (“FBS”) in a 96 wellplate for 36 hours before being treated with—kinase inhibitors or theantibodies at the specified concentration. Cell proliferation wasdetermined using the cell counting WST-8 kit (Dojindo technologies)following the manufacturer's instructions and using the formula: %survival rate=(Sample-Background)(Negative control-Background).Lapatinib was ground using a mortar and pestle and suspended in DMSO aspreviously described (Chen et al., Molecular cancer therapeutics 2012;11:660-9). To determine statistical significance, the results wereanalyzed using one-way ANOVA using Prism 6.0.

For T cell proliferation, naïve T cells were purified from human PBMCusing Pan T cell isolation kit (Miltenyi Biotec). 2×10⁵ purified T cellswere mixed with different antibodies in 96-well cell culture plate to afinal volume of 250 μl/well. T cells were cultured and maintained inRPMI-1640 supplemented with 10% FBS in 37° C. for 6 days. T cellproliferation was quantitated using the WST-8 kit as described above.

6.3.2.5 Cytotoxicity Assays (⁵¹Chromium Release Assay)

Cell cytotoxicity was assayed by ⁵¹Cr release as previously described(Xu et al., Cancer immunology research 2015; 3:266-77), and EC50 wascalculated using SigmaPlot software. Effector PBMC cells were purifiedfrom buffy coats purchased from the New York Blood Center. ATCs werefirst purified from human PBMC using Pan T cell isolation kit, and thenactivated and expanded for approximately 14 days with CD3/CD28 Dynabeads(Invitrogen) according to manufacturer's protocol. For pre-incubationexperiment, HER2-BsAb was pre-incubated with either ATCs (T cellspre-armed) or chromium-labeled tumor target cells (AU565 pre-targeted)for 30 minutes at room temperature, and unbound BsAb was washed off fortwo times before adding the other cells.

6.3.2.6 Cytokine Release Assay

Cytokine release was assayed as previously described (Ahmed et al.,OncoImmunology 2015; 4:e989776), using naïve T cells prepared asdescribed above. T cells (200,000/well) were cultured with or withoutNCI-N87 tumor cells (10,000/well) for 24 hours before supernatants beingharvested for ELISA-based cytokine assay.

6.3.2.7 PD-1/PD-L1 Expression

To overexpress PD-L1 in HEK293 cells, cells were cultured in DMEM(Cellgro) supplemented with 10% heat-inactivated FBS and Penicillin (100IU/ml) and streptomycin (100 μg/ml). HEK293 cells were plated into 6well plates at 0.5 million cells/well with 2 ml fresh media the daybefore transfection. Transfection was done with 2.5 μg hPD-L1 plasmidDNA using Lipofectamine 2000 (Invitrogen) according to manufacturer'sprotocol. Cells were incubated at 37° C. for 48 hours before harvestingwith 2 mM EDTA in PBS. 100,000-200,000 cells were used for FACS analysisand the rest were used for the killing assays.

To induce PD-1 expression in ATCs, effector cells were incubated in a3:1 ratio for 24 hours with the HER2(+) breast carcinoma cell lineHCC1954, after these target cells were incubated with HER2-BsAb at aconcentration of 10 μg/mL for 30 minutes and excessive antibody wasremoved. Cells were harvested and used in cytotoxicity assays aspreviously described against the HEK293 cells or HCC1954 cells. For PD-1blockade, PD-1-induced ATCs were pre-incubated with 10 μg/mLpembrolizumab for 30 minutes before adding to the well.

6.3.2.8 In Vivo Experiments

All animal procedures were performed in compliance with InstitutionalAnimal Care and Use Committee (IACUC) guidelines. For in vivo therapystudies, BALB-Rag2−/−IL-2R-γc-KO (DKO) mice (derived from colony of Dr.Mamoru Ito, CIEA, Kawasaki, Japan)(Koo et al., Expert Rev Vaccines 2009;8:113-20; Andrade et al., Arthritis Rheum 2011; 63:2764-73) were used.Four humanized mouse xenograft models were used: (1) intravenous(“i.v.”) tumor plus i.v. effector cells, (2) subcutaneous (“sc”) tumorplus sc effector cells, (3) sc tumor plus i.v. effector cells, and (4)intraperitoneal (“i.p.”) tumor plus i.p./i.v. effector cells. Patientderived xenografts (“PDXs”) were established from fresh surgicalspecimens with Memorial Sloan Kettering Cancer Center InstitutionalReview Board approval. Effector PBMC cells and ATCs were prepared asdescribed above. Prior to every experimental procedure, PBMCs and ATCswere analyzed by FACS for their percentage of CD3, CD4, CD8 and CD56cells to ensure consistency. Antibodies were injected i.v. or i.p. twicea week started two days before effectors cells for 3-6 weeks, given asi.v. 5-10×106 PBMC/ATC per injection, once a week for 2-3 weeks. s.c.xenografts were created with tumor cells suspended in Matrigel (CorningCorp, Tewksbury Mass.) and implanted in the flank of DKO mice. Tumorsize was measured using 1) hand-held TM900 scanner (Pieira, Brussels,BE), 2) Calipers, or 3) Bioluminescence. Bioluminescence imaging wasconducted using the Xenogen In Vivo Imaging System (IVIS) 200 (CaliperLifeSciences). Briefly, mice were injected i.v. with 0.1 mL solution ofD-luciferin (Gold Biotechnology; 30 mg/mL stock in PBS). Images werecollected 1 to 2 minutes after injection using the following parameters:a 10- to 60-second exposure time, medium binning, and an 8 f/stop.Bioluminescence image analysis was performed using Living Image 2.6(Caliper LifeSciences).

6.3.2.9 Immunohistochemistry Staining

The immunohistochemical detection was performed at Molecular CytologyCore Facility of Memorial Sloan Kettering using Discovery XT processor(Ventana Medical Systems). Paraffin-embedded tumor sections weredeparaffinized with EZPrep buffer (Ventana Medical Systems), antigenretrieval was performed with CCl buffer (Ventana Medical Systems) andsections were blocked for 30 minutes with Background Buster solution(Innovex). Anti-CD3 (DAKO, cat #A0452, 1.2 μg/ml), anti-HER2 (Enzo, cat#ALX-810-227, 5 μg/ml), and anti-PD-1 (Ventana, cat #760-4895, 3.1ug/ml) antibodies were applied and sections were incubated for 5 hours,followed by 60 minutes incubation with biotinylated goat anti-rabbit IgG(Vector labs, cat #PK6101) for CD3 and HER2 antibodies, or biotinylatedhorse anti-mouse IgG (Vector Labs, cat #MKB-22258) for PD-1 antibodiesat 1:200 dilution. The detection was performed with DAB detection kit(Ventana Medical Systems) according to manufacturer's instruction.Slides were counterstained with hematoxylin and coverslipped withPermount (Fisher Scientific). For PD-L1 staining, the sections werepre-treated with Leica Bond ER2 Buffer (Leica Biosystems) for 20 minutesat 100° C. The staining was done on Leica Bond RX (Leica Biosystems)with PD-L1 mouse monoclonal antibody (Cell Signaling, cat #29122, 2.5μg/ml) for 1 hour on Leica Protocol F. All images were captured fromtumor sections using Nikon ECLIPSE Ni-U microscope and NIS-Elements 4.0imaging software.

6.3.2.10 Statistics

Differences between samples indicated in the figures were tested forsignificance by one-way ANOVA using Prism 6.0, and p<0.05 was consideredstatistically significant.

6.3.3 Results 6.3.3.1 HER2-BsAb

HER2-BsAb heavy chain was constructed using the standard human IgG1,except for the N297A mutation in the Fc region to remove glycosylation.The light chain was constructed by extending the trastuzumab IgG1 lightchain with a C-terminal (G₄S)₃ linker followed by huOKT3 scFv (Xu etal., Cancer Immunology Research 2015; 3:266-77). The DNAs encoding bothheavy chain and light chain were inserted into a mammalian expressionvector, transfected into CHO-S cells, and stable clones of highestexpression were selected. Supernatants were collected from shaker flasksand purified on protein A affinity chromatography (Xu et al., CancerImmunology Research 2015; 3:266-77).

The SEC-HPLC and SDS-PAGE of the HER2-BsAb was analyzed. Under reducingSDS-PAGE conditions, HER2-BsAb gave rise to two bands at around 50 KDa,since the huOKT3 scFv fusion to trastuzumab light chain increased the MWto ˜50 KDa (data not shown). SEC-HPLC showed a major peak (97% by UVanalysis) with an approximate MW of 210 KDa, as well as a minor peak ofmultimers (data not shown). The BsAb remained stable by SDS-PAGE andSEC-HPLC after multiple freeze and thaw cycles (data not shown).

6.3.3.2 HER2-BsAb Retained Specificity, Affinity and Anti-ProliferativeEffects of Trastuzumab

To determine if HER2-BsAb retained the specificity of trastuzumab, theHER2(+)high SKOV3 ovarian carcinoma cell line was pre-incubated with 10μg/mL of trastuzumab for 30 minutes and then immunostained using 1 μg/mLHER2-BsAb labeled with Alexa 488 (FIG. 26A). Pre-incubation withtrastuzumab prevented HER2-BsAb from binding to SKOV3 cells,demonstrating that these antibodies shared the same specificity. Tocompare the avidity of HER2-BsAb to trastuzumab, the same cell line wasincubated with 10 fold downward dilutions (from 10 μg/ml to 1×10⁵ μg/mL)of trastuzumab or HER2-BsAb and analyzed by flow cytometry. The meanfluorescence intensity (“MFI”) was plotted against the antibodyconcentration in μM. Again the similarity in the binding curvesconfirmed that trastuzumab and HER2-BsAb had similar binding aviditiesfor their common HER2 target (FIG. 26B).

Finally, the trastuzumab-sensitive breast carcinoma cell line SKBR3 wastreated with Isotype control mAb, 10 nM lapatinib (as a positivecontrol), 10 μg/mL HER2-BsAb or 10 μg/mL trastuzumab for 72 hours andcell proliferation was assayed. As shown in FIG. 26C, trastuzumab andHER2-BsAb had similar anti-proliferative effects that were significantcompared to the negative control. As expected, lapatinib showed thestrongest inhibition of cell proliferation.

6.3.3.3 HER2-BsAb Redirected T Cell Cytotoxicity was HER2 Specific andDependent on CD3

Prior to the cytotoxicity assay, HER2-BsAb was shown capable of bindingdifferent T cells at the similar level (MFI around 450 with the BsAbconcentration of 1 ug/106 cells), no matter whether they were naïve Tcells purified from fresh PBMC or activated T cells (ATCs) (FIG. 27A).To establish the specificity of cytotoxic responses by T cells in thepresence of HER2-BsAb, HER2-negative (“HER2(−)”) breast carcinomaHTB-132 cells and HER2(+) MCF-7 cells were tested in a cytotoxicityassays using ATCs (E:T ratio of 10:1) and HER2-BsAb at decreasingconcentrations (FIG. 26D). Cytotoxicity was robust for HER2(+) cells butabsent for HER2(−) cells. In fact, HER2-BsAb was able to redirectefficient T cell killing no matter whether BsAb was present throughoutthe 4 hour assay (mixing), or used to pre-arm T cells and then washedoff, or to pre-target AU565 tumor cells and then washed off. Althoughpre-targeted AU565 cells were killed as well as mixing all threetogether, pre-armed T cells were less potent due to the low avidity ofBsAb binding to CD3 on T cells (FIG. 27B). To demonstrate the dependencyof cytotoxicity on both HER2 and CD3, HER2-BsAb cytotoxicity againstHER2(+) SCCHN cell line PCI-13 was tested in the presence oftrastuzumab, or the CD3 specific blocking huOKT3 IgG1 (FIG. 26E).Pre-incubation with either trastuzumab or huOKT3 prevented HER2-BsAbmediated T-cell cytotoxicity.

6.3.3.4 HER2-BsAb Mediated Cytotoxicity Against HER2(+) Cell Lines thatwere Resistant to Other HER2 Targeted Therapies.

A panel of a total of 39 cell lines from different tumor systems(breast, ovarian, gastric, head and neck, sarcoma, etc.) wascharacterized for their HER2 expression levels by flow cytometry and CTLactivity (Table 9). In this panel, 75% of these cells were testedpositive for HER2 expression. Representative cell lines were assayed fortheir sensitivity to tyrosine kinase inhibitors (10 nM each ofErlotinib, Lapatinib, Neratinib), or HER antibodies (10 μg/mL each oftrastuzumab and cetuximab), as well as HER2-BsAb mediated T-cellcytotoxicity. FIG. 28A, FIG. 28B and FIG. 28C showed threerepresentative lines from three different tumor systems. As shown, HER2expression, even in low quantities, was sufficient to mediate T-cellcytotoxicity in the presence of ATC and HER2-BsAb in cell linesotherwise resistant in in vitro to HER-targeted therapies. When thesecell lines were tested for cytotoxicity in the presence of ATC andHER2-BsAb, sensitivity to HER2-BsAb expressed as EC50 inverselycorrelated with surface HER2 expression in general (FIG. 28D, Table 9).

TABLE 9 HER2 Expres- sion EC50 Tumor Type Cell Line (MFl)* (pM)** Breastcarcinoma AU565 1175 0.3 Gastric Carcinoma NCI-N87 4900 1.1 OvarianCarcinoma OVCAR3 183 1.8 Breast Carcinoma MDA-MB-361 777 2.5 OvarianCarcinoma SKOV3 1577 2.8 Melanoma SKMEL28 190 3 Breast Carcinoma SKBR32506 4.1 Breast Carcinoma HCC1954 1597 5.5 Head and Neck Cancer SCC90274 5.7 Ewings SKEAW 246 10 Osteosarcoma CRL1427 108 10 RhabdomyosarcomaHTB82 204 10 Osteosarcoma RG 160 563 11 Head and Neck Cancer PCI-30 35912.2 Gastric Carcinoma KATO III 201 13.5 Melanoma HT-144 156 15Neuroblastoma NB5 66 15.5 Osteosarcoma RG 164 439 17.7 Head and NeckCancer UM SCC47 302 19.8 Osteosarcoma U2OS 90 22.5 GastricAdenocarcinoma AGS 172 23 Head and Neck Cancer UDSCC2 178 26.9 GastricCarcinoma SNU-16 29 30.5 Head and Neck Cancer 93VU147T 127 32.4 EwingsSKES-1 146 50 Breast Carcinoma MDA-MB-231 76 50.2 Head and Neck Cancer15B 305 62.8 Breast Carcinoma MCF7 398 64.9 Cervical Cancer HeLa 104120.7 Melanoma M14 57 130 Breast Carcinoma MDA-MB-468 6 >5000Neuroblastoma NMB7 12 >5000 Neuroblastoma SKNBE(2)C 8 >5000Neuroblastoma IMR32 6 >5000 Neuroblastoma SKNBE(2)S 4 >5000Neuroblastoma SKNBE(1)N 3 >5000 Small Cell lung Cancer NCI-H524 14 >5000Small Cell lung Cancer NCI-H69 10 >5000 Small Cell lung Cancer NCI-H3456 >5000

6.3.3.5 HER2-BsAb Mediated In Vitro T-Cell Cytotoxicity was RelativelyInsensitive to PD-L1 Expression on the Tumor Targets or PD-1 Expressionon T Cells.

Activation of tumor-specific CTL in the tumor microenvironment is knownto promote expression of PD-1/PD-L1, leading to T-cell exhaustion orsuppression, a phenomenon termed “adaptive immune resistance” (Tumeh etal., Nature 2014; 515:568-71). The presence of PD-1/PD-L1 pathway hasalso been reported to limit the anti-tumor effects of T-cell engagingbispecific antibodies (Junttila et al., Cancer Research 2014;74:5561-71). To determine if HER2-BsAb had this same limitation,PD-1-positive (“PD-1(+)”) ATCs were used against the HER2(+)PD-L1-positive (“PD-L1(+)”) breast carcinoma cell line HCC1954 with orwithout the PD-1-specific antibody pembrolizumab. As shown in FIG. 29A,PD-1(+) T cells generated similar cytotoxic responses in the presence ofHER2-BsAb no matter whether pembrolizumab was present or not. WhenHER2(+) human embryonic kidney cells (HEK-293) were transfected with thefull sequence of PD-L1 and used as targets, cytotoxicity against cellsexpressing PD-L1 was not significantly different to the cytotoxicityobserved in non-transfected HEK-293 cells, although maximal cytotoxicitywas slightly less with PD-L1(+) HEK-293 versus PD-L1-negative(“PD-L1(−)”) parental HEK-293 (FIG. 29B).

6.3.3.6 HER2-BsAb was Effective Against HER2(+) Xenografts

To determine the in vivo efficacy of HER2-BsAb, the breast carcinomacell lines HCC1954 (HER2high) and MCF-7 (HER2low), ovarian carcinomacell line SKOV3, and HER2(+) patient-derived breast cancer and gastriccancer xenografts (“PDXs”) were used in DKO mice xenograft models. Fourtumor models differing in tumor locations and effector routes were used,with the first three described before (Xu et al., Cancer ImmunologyResearch 2015; 3:266-77) to simulate different clinical situations: (1)intravenous (“i.v.”) tumor cells/i.v. effector PBMC; (2) subcutaneous(“s.c.” tumor cells/s.c. PBMC; (3) s.c. tumor cells/i.v. PBMC; and (4)intraperitoneal (“i.p.” tumor cells plus i.p. or i.v. effector T cellsto simulate ovarian cancer metastasizing to the peritoneal cavity. FIG.30 and FIG. 31 summarize the results of these experiments using celllines, and FIG. 32 summarizes the results of these experiments usingPDXs (M37 breast cancer and EK gastric cancer).

3×10⁶ HER2low MCF-7-luc (carrying luciferase reporter) cells wereinoculated via tail vein i.v. injection into DKO mice. When tumorpresence was confirmed by bioluminescence, mice were treated withHER2-BsAb or control BsAb (20 μg i.v., 2 times per week for 3 weeks), incombination with PBMC (5×10⁶ i.v., once per week for 2 weeks). Mice wereevaluated for tumor burden using luciferin bioluminescence every week.In this hematogenous disease model, MCF-7 cells were completelyeradicated without disease progression (FIG. 30A). This same cell linewas implanted s.c. mixed with effector PBMC (1:1, 7×10⁶ each), andtreated with HER2-BsAb (10 μg i.v., 2 times per week for 2 weeks in the1st experiment; or 20 μg i.v. 2 times per week for 3 weeks in the 2ndexperiment). In both experiments, HER2-BsAb caused a significant delayin tumor progression, while PBMC+trastuzumab or PBMC alone wasineffective (FIG. 30B). In two other separate experiments, 5×10⁶ HER2(+)breast carcinoma HCC1954 cells were implanted s.c. mixed with 2.5×10⁶PBMC (2:1). Again, after either 4 or 6 injections of HER2-BsAb (20 μgi.v. per dose), there was complete suppression of tumor growth, whiletrastuzumab or control BsAb HER2-C825 almost had no effect (FIG. 30C).In the third model, where s.c. 5×10⁶ HCC1954 xenografts were treatedwith i.v. PBMC (5×10⁶, once per week for 3 weeks) and i.v. HER2-BsAb(100 μg, twice per week for 3 weeks), tumor growth was substantiallydelayed (2 separate experiments), in contrast to only modest effects forPBMC+trastuzumab+huOKT3, PBMC+trastuzumab, or PBMC+huOKT3 (FIG. 30D).For HCC1954 xenografts, the following observations were made: (1) wheneffector PBMCs were mixed with tumor cells s.c., complete tumorregression without recurrence was seen past 90 days from tumorimplantation; and (2) when effector PBMCs were administered i.v., therewas significant reduction in the size of the tumors, but completeregression was only observed in a subset of animals (data not shown).

Since T cell homing into tumor is critical for anti-tumor response incancer immunotherapy (Tang et al., Cancer Cell 2016; 29:285-96), T-celltumor infiltration was studied using the s.c. tumor model described inFIG. 30D. Tumors were collected 5 days after i.v. PBMC andimmunohistochemistry (“IHC”) was performed (FIG. 30E). T-cell tumorinfiltration by CD3(+) staining was detected only inPBMC+HER2-BsAb-treated group, but not in control group(PBMC+Trastuzumab+huOKT3). These infiltrated T cells also had PD-1expression, although it was very weak. Interestingly, PD-L1 expressionin the tumor cells was strongly upregulated in the HER2-BsAb-treatedmice, presumably induced by the cytokines released by the infiltrated Tcells in the vicinity. But still, HER2-BsAb treatment eradicated thesetumors. This was consistent with the in vitro data (FIG. 29) showingHER2-BsAb-mediated T-cell cytotoxicity was relatively insensitive to orsufficient to overcome the PD-1/PD-L1 immune checkpoint inhibition.

To simulate ovarian cancer that metastasized into the peritoneal cavity,1×10⁵ ovarian cancer SKOV3-luc cells were injected peritoneally in DKOmice, and treatments were started after confirming tumor growth bybioluminescence. Besides i.v.ATC, i.p.ATC was also tested as a source ofeffectors. As shown in FIG. 31A and FIG. 31B, after treatment with ATC(7.5×10⁶ i.v. or i.p., once a week for 2 weeks), and i.p. HER2-BsAb (100μg, twice per week for 3 weeks), tumors were completely eradicatedwithout evidence of recurrence at followup. Both i.v.ATC and i.p.ATCwere equally effective in this fourth model.

HER2-BsAb was next tested using PDXs, since they could approximate thetumor heterogeneity and microenvironment typically found in fresh humantumor specimens. To determine whether HER2-BsAb is effective againstPDXs, two HER2(+) PDXs (gastric cancer PDX (EK) and breast cancer PDX(M37)) were tested using the s.c. tumor cells/i.v. PBMC model similar tothe one described in FIG. 30D. The PDXs were recently characterized byIHC using the PATHWAY anti-HER2/neu (4B5) Rabbit Monoclonal PrimiaryAntibody (VENTANA) and scored (according to the 2013 ASCO HER2 BreastCancer Testing Guidelines (see, e.g., Wolff et al., Journal of ClinicalOncology, 2013, 31(31):3997-4013)) as IHC 3+ for breast cancer PDX M37and IHC 2+ for gastric cancer PDX EK. When the gastric PDX (EK) waspassaged s.c. in DKO mice and treated with i.v. PBMC (1×10⁷, once a weekfor 3 weeks) and i.v. HER2-BsAb (100 μg, twice per week for 5 weeks),tumors were completely eradicated without disease progression, (FIG.32A), accompanied by substantial amount of T cell tumor infiltration(FIG. 32B), even though the HER2 expression level was relatively lowcompared to the M37 breast cancer PDX (FIG. 32C). In this nextexperiment, M37 PDX was passaged s.c. in DKO mice, and treated with i.v.PBMC (7.5×10⁶, once a week for 3 weeks) and i.v. HER2-BsAb (100 μg,twice per week for 6 weeks). Tumor growth was completely suppressed inthe group treated with HER2-BsAb and PBMC (FIG. 32D). Interestingly,despite characterization of the M37 PDX as IHC 3+, which is a HER2 levelindicative of suitability for treatment with trastuzumab (see, e.g.,Wolff et al., Journal of Clinical Oncology, 2013, 31(31):3997-4013),tumor growth was not suppressed in the M37 PDX group treated withPBMC+trastuzumab+huOKT3 (FIG. 32E). Taken together, these experimentsshowed that HER2-BsAb was effective against early passaged HER2(+) humantumor specimens.

6.3.4 Conclusions

This example described a HER2-specific BsAb with potent T cell mediatedanti-tumor activity in vitro and in vivo, ablating tumors or delayingtumor growth in four separate tumor-human PBMC compartment models.Unlike monovalent bispecifics, this HER2-BsAb had identicalanti-proliferative capacity to its parental trastuzumab. Its serumhalf-life and area under the curve were similar to IgG (data not shown).Most importantly, the T cell-mediated cytotoxicity it induced wasrelatively insensitive to inhibition by the PD-1/PD-L1 pathway, notpreviously described for this IgG-scFv platform (Xu et al., CancerImmunology Research 2015; 3:266-77). To date, other than the anti-GD2hu3F8-BsAb (Xu et al., Cancer Immunology Research 2015; 3:266-77), nopublished T-cell redirecting bispecific antibodies have used thisformat. The ability of this IgG-scFv antibody platform to recruitcirculating lymphocytes into the tumor stroma is critical, given theimportance of tumor-infiltrating lymphocyte (TIL) cells for a successfulanti-tumor effect in most checkpoint blockade studies to date (Tumeh etal., Nature 2014; 515:568-71), distinguishing responders fromnonresponders (Gajewski et al., Semin Oncol 2015; 42:663-71).

Schreiber has proposed that tumor cells evolve to evade the immunesystem through a process termed “immuno-editing”. Broadly speaking, thisprocess occurs at two levels: by changes within the (1) tumor cells or(2) the tumor microenvironment. Tumor cells can evade T-cell responsesby down-regulating MHC/peptide complexes or by decreasing tumor-antigenexpression or through the loss of antigen presenting machinerycomponents. On the other hand, suppression of the immune response in thetumor microenvironment is the result of T-regulatory cells,Myeloid-derived suppressor cells, M2 macrophages (Diaz-Montero et al.,Semin Oncol 2014; 41:174-84; Laoui et al., Frontiers in immunology 2014;5:489; Nishikawa & Sakaguchi, Curr Opin Immunol 2014; 27:1-7),immuno-suppressive cytokines (including IDO) (Munn & Mellor, TrendsImmunol 2013; 34:137-43), immune checkpoint molecules (Callahan et al.,Frontiers in Oncology 2014; 4:385; Postow et al., J Clin Oncol 2015;33(17):1974-82) and the consumption of IL-2 (Schreiber et al., Science2011; 331:1565-70).

Immune checkpoint antibodies that target the CTLA-4 and PD-1/PD-L1inhibitory pathways are capable of reversing the inhibitorytumor-microenvironment and producing significant and long-lastingclinical responses (Farolfi et al., Melanoma research 2012; 22:263-70).However, these strategies are not effective against all tumor types andtheir success is limited to a subset of patients. Durable clinicalresponses to the CTLA-4 blockade were recently correlated with tumormutational load and the expression of antigenic tetra-peptides thatresembled those found in viral and bacterial pathogens (Snyder et al., NEngl J Med 2014; 371:2189-99). Clonal neoantigens were shown to elicit Tcell immunoreactivity and sensitivity to blockade of the PD-1/PD-L1 axis(McGranahan et al., Science 2016; 351(6280):1463-9). Based on thesedata, the pre-existence of CD8(+) T-cells in the tumor (TILs) would becritical. More importantly, IHC evidence of negative regulation of tumorinfiltrating lymphocytes (TIL) by the PD-1/PD-L1 axis, was correlatedwith clinical response to checkpoint blockade (Tumeh et al., Nature2014; 515:568-71).

As data continues to accumulate, a consensus is emerging that theseimmune modulations would likely be ineffective against tumors with lowimmunogenicity because the presence of tumor specific lymphocytes isrequired for their clinical activity. Indeed, HER2 has been linked toimmune resistance (Seliger & Kiessling, Trends in Molecular Medicine2013; 19:677-84). This subset of patients, with “T-cell resistant”HER2(+) tumor cells and/or insufficient clonal frequency oftumor-specific T-cells, would likely not benefit from immune checkpointblockade alone. The unique property of the HER2-BsAb described in thisexample to recruit T-cells of any specificity and direct them againstestablished tumors with relative insensitivity to the PD-1 immunecheckpoint pathway is of interest, as it directly addresses the knownlimitations of immune checkpoint blockade. In fact, preliminary in vivodata showed no additional benefit of PD-1 blockade to the HER2-BsAbtherapeutic efficacy (data not shown), even though tumor PD-L1expression was up-regulated substantially following T cell infiltration(FIG. 30E).

When compared to the existing platforms that target HER2, HER2-BsAboffers advantages. Shalaby and colleagues described the development of abispecific (Fab′)2 antibody (anti-HER2 Fab′×anti-CD3 Fab′) throughexpressing each Fab′ separately and ligating the two together bychemical conjugation (Shalaby et al., J Exp Med 1992; 175:217-25). Morerecently, Junttila and colleagues developed a heterodimeric bispecificIgG (anti-HER2×anti-CD3) using “knob-and-hole” format (Junttila et al.,Cancer Research 2014; 74:5561-71). Both formats have monovalent bindingto either HER2 or CD3, and are substantially different, bothstructurally and functionally, when compared to the HER2-BsAb describedherein for the following reasons. First, the bivalent binding to HER2 iscritical for the anti-proliferation capability, which is preserved inthe HER2-BsAb construct described herein (FIG. 26C) but not in those twomonovalent systems, as demonstrated by Juntilla et al. Juntilla et al.showed that the anti-proliferation capability of monovalent binding toHER2 (either heterodimeric bispecific IgG or trastuzumab-Fab) was10-fold lower than bivalent trastuzumab (Junttila et al., CancerResearch 2014; 74:5561-71). Without being bound by any particulartheory, it is hypothesized that the dual mechanism (anti-proliferationplus T cell cytotoxicity) may create synergism and partly explains thepotent efficacy of the HER2-BsAb in vivo. This may provide a salvageoption for patients who progress on standard HER2-based therapies, or areplacement for trastuzumab given its dual anti-proliferative and T cellretargeting properties. Second, the bivalent binding to HER2 in our BsAbmaintains high avidity (FIG. 26B) so as to maximize tumor binding, whilethe monovalent binding to HER2 (either heterodimeric bispecific IgG ortrastuzumab-Fab) is 10-fold lower than trastuzumab (Junttila et al.,Cancer Research 2014; 74:5561-71). Higher avidity results in higher Tcell dependent cell cytotoxicity, a phenomenon that has beendemonstrated in T cell engaging bispecific antibodies (Ahmed et al.,OncoImmunology 2015; 4:e989776). Additionally, without being bound byany particular theory, it is hypothesized that the high avidity of theHER2-BsAb contributes to overcoming PD-1/PD-L1 checkpoints (FIG. 29),whereas the monovalent system by Juntilla was shown to be inhibited bythe PD-1/PD-L1 axis. Third, the BsAb described in this example has thetrastuzumab IgG backbone, preserving its pharmacologic advantages, whileShalaby's construct doesn't have FcR(n) affinity, should have muchshorter serum half-life, and probably needs to be administered as acontinuous infusion (as for Blinatumomab) to be effective in vivo.Fourth, the other advantage is manufacturability: once a CHO stable lineestablished, the HER2-BsAb can be produced in large scale and purifiedlike normal IgG without significant aggregation despite prolongedincubation at 37° C., while chemical conjugates require more complicatedsyntheses and downstream processing—each Fab′ expressed and purifiedseparately, chemically modified, and then the two chemically conjugatedand repurified. To ensure a final product that is pure and chemicallystable for direct clinical infusion is technically challenging andcostly. Such chemically crosslinked reagents have only been feasible forex vivo arming of T cells, but not for direct parenteral injections inthe clinic (Lum & Thakur A, BioDrugs 2011; 25:365-79).

A primary goal was to build a BsAb that has the bivalent binding totumor targets (to preserve high avidity and/or anti-proliferationcapability) and the monovalent binding to CD3 on effector T cells (tominimize spontaneous T cell activation in the absence of tumor targets).A number of uniquely different bivalent formats were surveyed, includingchemical conjugation (Yankelevich et al., Pediatr Blood Cancer 2012;59:1198-205), dual-variable-domain (DVD), or attaching huOKT3 scFv todifferent positions in the IgG backbone (C-terminal of heavy chain orC-terminal of light chain) (Kontermann, MAbs 2012; 4), and it was foundthat the last option gave the best functionality. Although the HER2-BsAbhas anti-CD3 scFv attached to both light chains, its reaction with CD3on T cells was considered as functionally monovalent primarily for thefollowing reasons. First, although the HER2-BsAb format contains twoanti-CD3 scFvs positioned at the end of the light chains, these scFvsare oriented in geometrically opposed directions which restrict theirability to cooperatively bind to neighboring CD3 on T cells. Thisrestricts the BsAb from binding bivalently and hence results infunctional monovalency to CD3. It has previously been shown in adifferent BsAb format that geometrical restriction of two anti-CD3 scFvcan result in functionally monovalent binding to T cell and lowercytokine release (Ahmed et al., OncoImmunology 2015; 4:e989776). Second,the functional consequence of bivalent binding to CD3 on T cells is thetriggering of spontaneous T cell activation, hence strong cytokinerelease in the absence of tumor targets. As shown in FIG. 33A, HER2-BsAbonly stimulated background cytokine release similar to that of themonovalent huOKT3 Fab, while bivalent huOKT3 IgG induced substantiallymore cytokines in the absence of tumor targets (left panel). However, inthe presence of HER2(+) NCI-N87 tumor target, anti-tumor TH 1 cytokines(TNFα and IFNγ) were released but only in the presence of BsAb (rightpanel), a format previously shown to induce immunologic synapseformation between the T cells and tumor targets (Xu et al., CancerImmunology Research 2015; 3:266-77). Furthermore, only bivalent huOKT3IgG induced robust T cell proliferation, while HER2-BsAb and monovalenthuOKT3 Fab had negligible effects comparable to the T cells only Control(FIG. 33B). In addition, the aglycosylation of the Fc removed both ADCCand most CMC functions, thereby further reducing cytokine releasewithout affecting serum pharmacokinetics or compromising T cellactivation.

Chimeric antigen receptor technology has rapidly acceleratedinvestigations into HER2-directed gene modified T cells in severalclinical trials: NCT00902044 (for sarcoma), NCT00889954 (all HER2(+)cancers), NCT01109095 (GBM), NCT00924287 (metastatic cancer), andNCT01935843 (HER2(+) solid tumors). Toxicities from off target effectswere initially concerning (Morgan et al., Mol Ther 2010; 18:843-51),although subsequent patients have been safely managed pharmacologically.There, the ability of T cells to overcome low levels of antigenexpression was again observed. Osteosarcoma was a good example where theexpression level has been controversial (Thomas et al., Clin Cancer Res2002; 8:788-93), and where CAR-modified T cells were highly efficientagainst locoregional and metastatic xenografts (Ahmed et al., Mol Ther2009; 17:1779-87), and against osteosarcoma tumor initiating cells(Rainusso et al., Cancer Gene Ther 2012; 19:212-7). Although thesuccessful clinical application of CAR T cells has reassured manyskeptics, there remain obstacles, including the necessity ofcytoreductive chemotherapy prior to T cell infusion for meaningfulclinical responses, logistics of cell harvest, processing, storage,transport and product release, T cell exhaustion (Long et al., Nat Med2015; 21:581-90) and inadequate T cell persistence after infusion.

In summary, this example demonstrates a successful IgG-scFv platform toengage T cells for HER2-directed immunotherapy. This BsAb forretargeting T cells was built with structural considerations forbivalency towards the target, and functionally monovalency towards CD3on effector T cells, plus Fc aglycosylation for minimal spontaneouscytokine release. Its relative insensitivity to the PD-1/PD-L1 axis wasnovel. It has excellent anti-tumor activity both in vitro and in vivo,which is superior to trastuzumab.

6.4 Example 4

This example demonstrates that HER2-BsAb described in section 6.3.3.1above (comprising a heavy chain consisting of the amino acid sequenceset forth in SEQ ID NO: 62 and a light chain fusion polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 60) iseffective against HER2(+) breast cancer cell line xenografts thatexpress PDL1 but are resistant to PD1 or PDL1 treatment (FIG. 34). SeeSection 6.3.2.8 for materials and methods. In particular, 5×10⁶ HCC1954xenografts were implanted subcutaneously in mice and mice were treatedintravenously with 7.5×10⁶ PBMC once per week for two weeks andintravenously with HER2-BsAb, anti-PD1 antibody Pembrolizumab, oranti-PDL1 antibody Atezolizumab. HER2-BsAb, anti-PD1 antibody, andanti-PDL1 antibody treatments were performed with 100 μg each, twice perweek for 4 weeks. HER2-BsAb-treated tumors were completely eradicated.In contrast, there was no effect on tumors treated with PD1/PDL1blockade (i.e., treatment with anti-PD1 antibody Pembrolizumab oranti-PDL1 antibody Atezolizumab).

7. EQUIVALENTS

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

1.-78. (canceled)
 79. A method of treating a HER2-positive cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a bispecific binding moleculecomprising an aglycosylated monoclonal antibody that is animmunoglobulin that binds to HER2, said immunoglobulin comprising twoidentical heavy chains and two identical light chains, said light chainsbeing a first light chain and a second light chain, wherein the firstlight chain is fused to a first single chain variable fragment (scFv),via a peptide linker, to create a first light chain fusion polypeptide,and wherein the second light chain is fused to a second scFv, via apeptide linker, to create a second light chain fusion polypeptide,wherein the first and second scFv (i) are identical, and (ii) bind toCD3, and wherein the first and second light chain fusion polypeptidesare identical, and wherein the cancer expresses a low level of HER2, isresistant to trastuzumab, and is selected from the group consisting ofbreast cancer, gastric cancer, an osteosarcoma, desmoplastic small roundcell cancer, ovarian cancer, prostate cancer, pancreatic cancer,glioblastoma multiforme, gastric junction adenocarcinoma,gastroesophageal junction adenocarcinoma, cervical cancer, salivarygland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma,rhabdomyosarcoma, or neuroblastoma.
 80. The method of claim 79, whereinthe cancer expresses HER2 levels that are lower relative to cancers thatare indicated for treatment with trastuzumab and are of the same tissuetype as the cancer.
 81. The method of claim 79, wherein the cancer hasbeen determined not to overexpress HER2 based on the followingcharacterization of the cancer: (a) a first determination of a level ofHER2 in a test specimen comprising cells of the cancer is reported asnegative, or (b) a first determination of a level of HER2 in a testspecimen comprising cells of the cancer is reported as equivocal, and asecond determination of a level of HER2 in a test specimen comprisingcells of the cancer is reported as equivocal or negative.
 82. The methodof claim 81, wherein (a) a determination of the level of HER2 in thetest specimen is reported as negative when the level of HER2 in the testspecimen is characterized as (i) (1) immunohistochemistry (IHC)1+,wherein the level of HER2 in the test specimen is characterized as IHC1+ when the test specimen exhibits an incomplete HER2 membrane stainingthat is faint/barely perceptible and within greater than 10% of theinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; (2) IHC 0, wherein the level of HER2 in the testspecimen is characterized as IHC 0 when the test specimen exhibits noHER2 staining observed, wherein the lack of staining is readilyappreciated using a low-power objective, or a HER2 membrane stainingthat is incomplete and is faint/barely perceptible and within less thanor equal to 10% of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; or (ii) in situhybridization (ISH) negative, wherein the level of HER2 in the testspecimen is characterized as ISH negative when the test specimenexhibits (1) a single-probe average HER2 copy number of less than 4.0signals per cell; or (2) a dual-probe HER2/CEP17 ratio of less than 2.0with an average HER2 copy number of less than 4.0 signals per cell, and(b) a determination of the level of HER2 in the test specimen isreported as equivocal when the level of HER2 in the test specimen ischaracterized as: (i) IHC 2+, wherein the level of HER2 in the testspecimen is characterized as IHC 2+ when the test specimen exhibits (1)a circumferential HER2 membrane staining that is incomplete and/orweak/moderate and within greater than 10% of invasive tumor cells,wherein the staining is observed in a homogenous and contiguouspopulation, and wherein the staining is readily appreciated using alow-power objective; or (2) a complete and circumferential HER2 membranestaining that is intense and within less than or equal to 10% ofinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; or (ii) ISH equivocal, wherein the level of HER2in the test specimen is characterized as ISH equivocal when the testspecimen exhibits (1) a single-probe ISH average HER2 copy number ofgreater than or equal to 4.0 and less than 6.0 signals/cell, wherein thecopy number is determined by counting at least 20 cells within the areaand is observed in a homogenous and contiguous population; or (2) adual-probe HER2/CEP17 ratio of less than 2.0 with an average HER2 copynumber of greater than or equal to 4.0 and less than 6.0 signals percell, wherein the copy number is determined by counting at least 20cells within the area and is observed in a homogenous and contiguouspopulation.
 83. The method of claim 79, wherein HER2 levels in a testspecimen comprising cells of the cancer is characterized as IHC 2+ orless according to applicable American Society of ClinicalOncology/College of American Pathologists guideline recommendations forhuman epidermal growth factor receptor 2 testing in cancer.
 84. Themethod of claim 83, wherein: (I) the level of HER2 in the test specimenis characterized as IHC 2+ when the test specimen exhibits (1) acircumferential HER2 membrane staining that is incomplete and/orweak/moderate and within greater than 10% of invasive tumor cells,wherein the staining is observed in a homogenous and contiguouspopulation, and wherein the staining is readily appreciated using alow-power objective; or (2) a complete and circumferential HER2 membranestaining that is intense and within less than or equal to 10% ofinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; (II) the level of HER2 in the test specimen ischaracterized as IHC 1+ when the test specimen exhibits an incompleteHER2 membrane staining that is faint/barely perceptible and withingreater than 10% of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; or (III) the level ofHER2 in the test specimen is characterized as IHC 0 when the testspecimen exhibits no HER2 staining observed, wherein the lack ofstaining is readily appreciated using a low-power objective, or a HER2membrane staining that is incomplete and is faint/barely perceptible andwithin less than or equal to 10% of the invasive tumor cells, whereinthe staining is readily appreciated using a low-power objective.
 85. Themethod of claim 79, wherein the cancer is a programmed death-ligand 1(PDL1)-positive cancer.
 86. The method of claim 85, wherein the canceroverexpresses PDL1 relative to expression of PDL1 in analogousnoncancerous cells of the same tissue type as the cancer.
 87. The methodof claim 85, wherein the cancer is resistant to PDL1 blockade with ananti-PDL1 therapy or PD1 blockade with an anti-PD1 therapy.
 88. Themethod of claim 87, wherein the anti-PDL1 therapy comprises atezolizumabor wherein the anti-PD1 therapy comprises pembrolizumab.
 89. The methodof claim 79, wherein: (a) the sequence of each heavy chain is any of SEQID NOs: 23, 27, 62 or 63; (b) the sequence of each light chain is SEQ IDNO: 25; (c) the sequence of the peptide linker is any of SEQ ID NOs: 14or 35-41; (d) the sequence of a V_(H) domain in the first scFv is any ofSEQ ID NOs: 15, 17 or 64; (e) the sequence of an intra-scFv peptidelinker between a V_(H) domain and a V_(L) domain in the first scFv isany of SEQ ID NOs: 14 or 35-41; (f) the sequence of a V_(L) domain inthe first scFv is any of SEQ ID NOs: 16 or 65; (g) the sequence of thescFv is any of SEQ ID NOs: 19 or 48-59; and/or (h) the sequence of thefirst light chain fusion polypeptide is any of SEQ ID NOs: 29, 34,42-47, or
 60. 90. The method of claim 79, wherein the sequence of eachheavy chain is SEQ ID NO: 27, and wherein the sequence of each lightchain is SEQ ID NO: 25; the sequence of the heavy chain is SEQ ID NO: 62and wherein the sequence of each light chain fusion polypeptide is SEQID NO: 60; the sequence of the heavy chain is SEQ ID NO: 27 and whereinthe sequence of each light chain fusion polypeptide is SEQ ID NO: 34;the sequence of the heavy chain is SEQ ID NO: 27 and wherein thesequence of each light chain fusion polypeptide is SEQ ID NO: 47; or thesequence of the heavy chain is SEQ ID NO: 27 and wherein the sequence ofeach light chain fusion polypeptide is SEQ ID NO:
 29. 91. The method ofclaim 79, wherein the HER2-positive cancer is resistant to treatmentwith cetuximab, lapatinib, erlotinib, or any other small molecule orantibody that targets the HER family of receptors.
 92. A method oftreating a HER2-positive cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of Tcells bound to a bispecific binding molecule, wherein the bispecificbinding molecule comprises an aglycosylated monoclonal antibody that isan immunoglobulin that binds to HER2, said immunoglobulin comprising twoidentical heavy chains and two identical light chains, said light chainsbeing a first light chain and a second light chain, wherein the firstlight chain is fused to a first single chain variable fragment (scFv),via a peptide linker, to create a first light chain fusion polypeptide,and wherein the second light chain is fused to a second scFv, via apeptide linker, to create a second light chain fusion polypeptide,wherein the first and second scFv (i) are identical, and (ii) bind toCD3, and wherein the first and second light chain fusion polypeptidesare identical, and wherein the cancer expresses a low level of HER2, isresistant to trastuzumab, and is selected from the group consisting ofbreast cancer, gastric cancer, an osteosarcoma, desmoplastic small roundcell cancer, ovarian cancer, prostate cancer, pancreatic cancer,glioblastoma multiforme, gastric junction adenocarcinoma,gastroesophageal junction adenocarcinoma, cervical cancer, salivarygland cancer, soft tissue sarcoma, leukemia, melanoma, Ewing's sarcoma,rhabdomyosarcoma, or neuroblastoma.
 93. The method of claim 92, wherein:(a) the sequence of each heavy chain is any of SEQ ID NOs: 23, 27, 62 or63; (b) the sequence of each light chain is SEQ ID NO: 25; (c) thesequence of the peptide linker is any of SEQ ID NOs: 14 or 35-41; (d)the sequence of a V_(H) domain in the first scFv is any of SEQ ID NOs:15, 17 or 64; (e) the sequence of an intra-scFv peptide linker between aV_(H) domain and a V_(L) domain in the first scFv is any of SEQ ID NOs:14 or 35-41; (f) the sequence of a V_(L) domain in the first scFv is anyof SEQ ID NOs: 16 or 65; (g) the sequence of the scFv is any of SEQ IDNOs: 19 or 48-59; and/or (h) the sequence of the first light chainfusion polypeptide is any of SEQ ID NOs: 29, 34, 42-47, or
 60. 94. Themethod of claim 92, wherein the sequence of each heavy chain is SEQ IDNO: 27, and wherein the sequence of each light chain is SEQ ID NO: 25;the sequence of the heavy chain is SEQ ID NO: 62 and wherein thesequence of each light chain fusion polypeptide is SEQ ID NO: 60; thesequence of the heavy chain is SEQ ID NO: 27 and wherein the sequence ofeach light chain fusion polypeptide is SEQ ID NO: 34; the sequence ofthe heavy chain is SEQ ID NO: 27 and wherein the sequence of each lightchain fusion polypeptide is SEQ ID NO: 47; or the sequence of the heavychain is SEQ ID NO: 27 and wherein the sequence of each light chainfusion polypeptide is SEQ ID NO:
 29. 95. The method of claim 92, whereinthe cancer expresses HER2 levels that are lower relative to cancers thatare indicated for treatment with trastuzumab and are of the same tissuetype as the cancer.
 96. The method of claim 92, wherein the cancer hasbeen determined not to overexpress HER2 based on the followingcharacterization of the cancer: (a) a first determination of a level ofHER2 in a test specimen comprising cells of the cancer is reported asnegative, or (b) a first determination of a level of HER2 in a testspecimen comprising cells of the cancer is reported as equivocal, and asecond determination of a level of HER2 in a test specimen comprisingcells of the cancer is reported as equivocal or negative.
 97. The methodof claim 96, wherein (a) a determination of the level of HER2 in thetest specimen is reported as negative when the level of HER2 in the testspecimen is characterized as (i) (1) immunohistochemistry (IHC) 1+,wherein the level of HER2 in the test specimen is characterized as IHC1+ when the test specimen exhibits an incomplete HER2 membrane stainingthat is faint/barely perceptible and within greater than 10% of theinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; (2) IHC 0, wherein the level of HER2 in the testspecimen is characterized as IHC 0 when the test specimen exhibits noHER2 staining observed, wherein the lack of staining is readilyappreciated using a low-power objective, or a HER2 membrane stainingthat is incomplete and is faint/barely perceptible and within less thanor equal to 10% of the invasive tumor cells, wherein the staining isreadily appreciated using a low-power objective; or (ii) in situhybridization (ISH) negative, wherein the level of HER2 in the testspecimen is characterized as ISH negative when the test specimenexhibits (1) a single-probe average HER2 copy number of less than 4.0signals per cell; or (2) a dual-probe HER2/CEP17 ratio of less than 2.0with an average HER2 copy number of less than 4.0 signals per cell, and(b) a determination of the level of HER2 in the test specimen isreported as equivocal when the level of HER2 in the test specimen ischaracterized as: (i) IHC 2+, wherein the level of HER2 in the testspecimen is characterized as IHC 2+ when the test specimen exhibits (1)a circumferential HER2 membrane staining that is incomplete and/orweak/moderate and within greater than 10% of invasive tumor cells,wherein the staining is observed in a homogenous and contiguouspopulation, and wherein the staining is readily appreciated using alow-power objective; or (2) a complete and circumferential HER2 membranestaining that is intense and within less than or equal to 10% ofinvasive tumor cells, wherein the staining is readily appreciated usinga low-power objective; or (ii) ISH equivocal, wherein the level of HER2in the test specimen is characterized as ISH equivocal when the testspecimen exhibits (1) a single-probe ISH average HER2 copy number ofgreater than or equal to 4.0 and less than 6.0 signals/cell, wherein thecopy number is determined by counting at least 20 cells within the areaand is observed in a homogenous and contiguous population; or (2) adual-probe HER2/CEP17 ratio of less than 2.0 with an average HER2 copynumber of greater than or equal to 4.0 and less than 6.0 signals percell, wherein the copy number is determined by counting at least 20cells within the area and is observed in a homogenous and contiguouspopulation.
 98. The method of claim 92, wherein the T cells that areautologous or allogeneic to the subject to whom the T cells areadministered.